Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations

Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.     

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.     

Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells.     

Manipulation of intracellular magnesium levels in Saccharomyces cerevisiae with deletion of magnesium transporters

Magnesium is an important divalent ion for organisms. There have been a number of studies in vitro suggesting that magnesium affects enzyme activity. Surprisingly, there have been few studies to determine the cellular mechanism for magnesium regulation. We wished to determine if magnesium levels could be regulated in vivo. It is known that Saccharomyces cerevisiae has two magnesium transporters (ALR1 and ALR2) across the plasma membrane. We created S. cerevisiae strains with deletion of one (alr1 or alr2) or both (alr1 alr2) transporters. The deletion of ALR1 resulted in a decrease in intracellular magnesium levels. An increase from 5 to 100 mM in the exogenous magnesium level increased the intracellular levels of magnesium in the alr1 and alr1 alr2 strains, whereas the expression of magnesium transporters from S. cerevisiae or Arabidopsis thaliana led to a change of the intracellular levels of magnesium in those strains. The deletion of magnesium transporters in A. cerevisiae and overexpression of magnesium transporters from A. thaliana also affected the intracellular concentrations of a range of metal ions, which suggests that cells use non-specific transporters to help regulate metal homeostasis.     

NAD(P)H oxidase and renal epithelial ion transport

A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na(+) and K(+) transport. Finally, we will review the relationship between tubular H(+) efflux and NADPH oxidase activity.     

Effect of high magnesium ion concentration on the electron transport rate and proton exchange in thylakoid membranes in higher plants]

The effects of magnesium ion concentration on the rate of electron transport in isolated pea thylakoids were investigated in the pH range from 4.0 up to 8.0. In the absence of magnesium ions in the medium and in the presence of 5 mM MgCl2 in the experiments not only without added artificial acceptors but also with ferricyanide or methylviologen as an acceptor, this rate had a well-expressed maximum at pH 5.0. It was shown that, after depression to minimal values at pH 5.5-6.5, it gradually rose with increasing pH. An increase in magnesium ion concentration up to 20 mM essentially affected the electron transfer rate: it decreased somewhat at pH 4.0-5.0 but increased at higher pH values. At this magnesium ion concentration, the maximum rate was at pH 6.0-6.5 and the minimum, at pH 7.0. Subsequent rise upon increasing pH to 8.0 was expressed more sharply. The influence of high magnesium ion concentration on the rate of electron transport was not observed in the presence of gramicidin D. It was found that without uncoupler, the changes in the electron transfer rate under the influence of magnesium ions correlated to the changes in the first-order rate constant of the proton efflux from thylakoids. It is supposed that the change in the ability of thylakoids to keep protons by the action of magnesium ions is the result of electrostatic interactions of these ions with the charges on the external surface of membranes. A possible role of regulation of the electron transport rate by magnesium ions in vivo is discussed.     

The Effects of Probenecid and Thiazides and Their Combination on the Urinary Excretion of Electrolytes and on Acid-base Equilibrium

The effects of commonly used therapeutic doses of hydrochlorothiazide and probenecid, given singly and in combination, on the urinary excretion of monovalent and divalent ions and on acid-base equilibrium were studied in four patients with idiopathic hypercalciuria.Probenecid had no effect on the urinary excretion of monovalent ions but resulted in a sustained increase in the urinary excretion of calcium, magnesium and citrate and a temporary increase in the urinary excretion of ammonium, in addition to its well-known effects on uric acid metabolism. A temporary fall in serum phosphorus levels was also observed.Probenecid also modified the response to hydrochlorothiazide in that the urinary excretion of calcium, magnesium and citrate was greater during combined therapy than when hydrochlorothiazide was administered alone. Probenecid prevented or abolished the increase in serum uric acid levels associated with the use of thiazide but did not modify the effects of hydrochlorothiazide on the urinary excretion of sodium, chloride, potassiu, phosphorus, ammonium, titratable acid and bicarbonate.     

Tauroursodeoxycholate prevents taurocholate induced cholestasis

The nature of transport pathway(s) for the biliary excretion of taurocholate and tauroursodeoxycholate was examined by comparing the biliary transport maximum (Tm) value for taurocholate during the infusion of taurocholate alone with that of taurocholate combined with tauroursodeoxycholate. The combined infusion of tauroursodeoxycholate resulted in an appreaciable excretion of tauroursodeoxycholate while the excretion rate of taurocholate was not reduced in comparison with the Tm value of taurocholate alone. Furthermore, the Tm state of taurocholate was maintained for a much longer period with the simultaneous infusion of tauroursodeoxycholate than by the infusion of taurocholate alone. The cholestasis usually produced by the excess infusion of taurocholate was also prevented when tauroursodeoxycholate was simultaneously infused. Since plasma taurocholate concentration was not significantly different between the two rat groups, the results suggest the presence of the facilitative interaction of tauroursodeoxycholate with the taurocholate excretion.     

Tubular Transport Maxima of PAH and Diodrast Measured Individually in the Aglomerular Kidney of Lophius, and Simultaneously as Competitors Under Conditions of Equimolar Loading

The maximal tubular transfer rates (Tm) of both p-aminohippurate (PAH) and diodrast (3,5-diiodo-4-pyridone-N-acetic acid or iodopyracet) were found to be fixed and reproducible when measured separately in Lophius (goosefish) under standard laboratory conditions. Expressed on a molar basis Tm_PAH was four times Tm_D. However, when these transport competitors were presented simultaneously in equimolar concentrations with the plasma levels of each sufficiently high enough to saturate the carrier system, the relative rates of excretion were reversed with the diodrast transfer rate then four times that of PAH. The combined rate of excretion was far below Tm_PAH alone, and roughly equal to Tm_D. Interaction with a common carrier was indicated by the gradations in degree of inhibition which resulted when plasma concentration ratios of diodrast to PAH were extended from 0.1 to 3.2, and PAH transfer rates expressed as percentage of Tm_PAH were correspondingly depressed from 17 to 1.0 per cent respectively. These observations again point up the inverse relationship between transfer rate and competitive effectiveness which exists for members of a series of substances actively transported by a common mechanism. It appears that carrier affinity and dissociation characteristics may be quite different for various compounds in a series, and also that these parameters may vary significantly from species to species.     

Stimulation of renal phosphate secretion in the stenohaline freshwater teleost: Carassius auratus gibelio Bloch

1. Renal excretion of phosphate in the Prussian carp was modulated by tubular reabsorption and tubular secretion. 2. Under the conditions of an i.v. phosphate-load during which the plasma concentration of phosphate was doubled, 65% of the phosphate load was excreted by the kidney, mainly by tubular secretion. 3. The renal clearance of PAH markedly exceeded the clearance of polyfructosan which could be used for the measurement of the GFR. 4. A transport maximum for tubular PAH secretion was reached at plasma concentrations of about 250 mumol/l.     

Effects of a prostacyclin analog iloprost on kidney function, renin-angiotensin and kallikrein-kinin systems, prostanoids and catecholamines in man

Iloprost (ZK 36 374), a stable analog of carbaprostacyclin, was infused for 72 h to nine patients with advanced obliterative arterial disease. Iloprost caused a marked vasodilation and a compensatory increase in cardiac output. The glomerular filtration rate increased by 45% and tubular reabsorption of sodium and water were reduced by 80% and 107%, respectively. The urine excretion rate increased by 122%. Tubular handling of potassium and calcium were not influenced by iloprost but magnesium reabsorption was stimulated. The renin-angiotensin system was not activated while serum angiotensin converting enzyme activity was decreased. Kallikrein excretion in urine was increased 4.4-fold but plasma kininogen, a substrate for kallikrein in producing vasoactive kinins, was unaffected by the drug. Plasma levels of 6-keto-PGF1 alpha and TxB2 were decreased and their excretion in urine increased. Plasma catecholamines were not changed by iloprost. Several of the changes persisted for at least the first postinfusion day. The results indicate that iloprost increases urine excretion rate by increasing glomerular blood flow and by inhibiting sodium and water reabsorptions. The kinin-forming system, but not the renin-angiotensin system or plasma catecholamines, may be activated. The decrease in plasma level of prostanoids can be, at least partly, due to their increased excretions in urine.     

UPTAKE OF MANGANESE BY CHROMAFFIN GRANULES IN VITRO

Abstract– The distribution of metal ions in the chromaffin granule is anomalous. Calcium ions are accumulated but magnesium ions excluded. We have used the manganese ion as a convenient divalent element for following metal ion uptake because it can usefully be employed as a substitute for magnesium.
The kinetics of the manganese incorporation were followed by radio tracer technique and its transport was characterized as being non-energy dependent and having a small thermal activation factor. The presence in the incubation medium of a membrane-bound ATPase inhibitor (NEM) or addition of CN⁻ or NaN₃ did not affect the metal incorporation into chromaffin granules. Little endogenous magnesium exchanges for manganese when the latter is incorporated, but magnesium competitively prevented manganese uptake. The binding of manganese inside the vesicle was strong and it was not exchangeable with magnesium even in the presence of ATP.
Manganese when incorporated binds to ATP and displaces catecholamines resulting in a decrease in the CA/ATP ratio.     

Tubular secretion of creatine,trimethylamine oxide,and other organic bases by the aglomerular kidney of Lophius americanus

Creatine and trimethylamine oxide (TMAO) are the chief nitrogenous constituents of normal Lophius urine, and both of these organic bases characteristically have high urine/plasma concentration ratios. Competition studies involving various organic bases indicate that creatine and TMAO are excreted independently by separate transport mechanisms. TMAO excretion is inhibited competitively by tetraethylammonium ion (TEA) and by cyanine dye No. 863—compounds previously shown to be transferred actively by an organic base-secreting mechanism of general occurrence among vertebrates. TEA does not inhibit competitively the active tubular reabsorption of TMAO in Squalus with doses which markedly depress its tubular excretion in Lophius. Glycine, which inhibits creatine reabsorption in the dog, does not interfere competitively with its secretion in Lophius.     

TRPM6 and TRPM7--Gatekeepers of human magnesium metabolism

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems - next to passive paracellular magnesium flux - involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.     

TRPM6 and TRPM7—Gatekeepers of human magnesium metabolism

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems – next to passive paracellular magnesium flux – involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.     

Characterization of an integral protein of the brush border membrane mediating the transport of divalent metal ions

The transport of Fe(2+) and other divalent transition metal ions across the intestinal brush border membrane (BBM) was investigated using brush border membrane vesicles (BBMVs) as a model. This transport is an energy-independent, protein-mediated process. The divalent metal ion transporter of the BBM is a spanning protein, very likely a protein channel, that senses the phase transition of the BBM, as indicated by a break in the Arrhenius plot. The transporter has a broad substrate range that includes Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Under physiological conditions the transport of divalent metal ions is proton-coupled, leading to the acidification of the internal cavity of BBMVs. The divalent metal ion transporter can be solubilized in excess detergent (30 mM diheptanoylphosphatidylcholine or 1% Triton X-100) and reconstituted into an artificial membrane system by detergent removal. The reconstituted membrane system showed metal ion transport characteristics similar to those of the original BBMVs. The properties of the protein described here closely resemble those of the proton-coupled divalent cation transporter (DCT1, Nramp2) described by, Nature. 388:482-488). We may conclude that a protein of the Nramp family is present in the BBM, facilitating the transport of Fe(2+) and other divalent transition metal ions.     

The role of hydrated divalent metal ions in the bridging of two anionic groups. An ab initio quantum chemical and molecular mechanics study of dimethyl phosphate and formate bridged by calcium and magnesium ions

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.     

Effects of cobalt, magnesium, and cadmium on contraction of rat soleus muscle.

The effects on isometric tension of three divalent ions that block calcium channels, magnesium, cobalt, and cadmium, were tested in small bundles of rat soleus fibers. Cobalt, at a concentration of 2 or 6 mM, reversibly depressed twitch and tetanic tension and the depression was much greater in solutions containing no added calcium ions. Magnesium caused much less depression of tension than cobalt. The depression of tension was not accompanied by membrane depolarization or a reduction in the amplitude of action potentials. A reduction caused by 6 mM cobalt in the amplitude of 40 or 80 mM potassium contractures was not accompanied by a comparable reduction in tension during 200 mM potassium contractures, and could be explained by a shift in the potassium contracture tension-voltage curve to more positive potentials (by +7 mV on average). Similar effects were not seen with 2 or 6 mM magnesium. At a concentration of 20 mM, both cobalt and magnesium depressed twitch and tetanic tension, cobalt having greater effect than magnesium. Both ions shifted the potassium contracture tension-voltage curve to the right by +5 to +10 mV, caused a small depression of maximum tension, and slowed the time course of potassium contractures. Cadmium (3 mM) depressed twitch, tetanic, and potassium contracture tension by more than 6 mM cobalt, but experiments were complicated by the gradual appearance of large contractures that became even larger, and sometimes oscillatory, when the solution containing cadmium was washed out. It was concluded that divalent cations affect both activation and inactivation of tension in a manner that cannot be completely explained by a change in surface charge.     

Influence of ions and tonicity of RNA metabolism in Tetrahymena pyriformis

Net RNA degradation occurs in Tetrahymem pyrifmmis when this ciliate is suspended in a non-nutrient medium. The quantity and quality of the excretion products is at least partially under the control of the ionic content and the tonicity of the cellular environment. The excretion of ultraviolet-absorbing materials was found to be elevated by sodium ions in a medium isotonic to the culture fluid, or by a hypertonic environment. Magnesium counteracted these effects. In isotonic suspension, sodium and magnesium ions lowered orthophosphate excretion; however, sodium altered the nature of the phosphate products so that acidlabile phosphates were also excreted rather than solely orthophosphate. Similar results were obtained in a hypertonic environment with or without sodium. The degree of purine and pyrimidine loss from the cells in all conditions of suspension was reflected in the amount of RNA degraded. The ion and tonicity effects apparently reflect events which alter the stability of the RNA and the properties of the membrane system, resulting in changes in both the rate of RNA degradation and the nature of the excreted products. The rates of orthophosphate excretion appear to be affected by changes in the acid-base balance within the cell which may be governed by the cation levels. The manipulation of the ionic content and tonicity of the medium offers a convenient method for obtaining cells reduced in RNA content.     

Water pH and urinary excretion in silver catfish Rhamdia quelen

The effect of acute exposure to different water pH levels on urinary excretion and plasma ion levels in silver catfish Rhamdia quelen was analysed. Fish were exposed to pH 4·0, 5·0, 7·5, 8·0, and 9·0 for 4 days and urine was collected. Other specimens were also exposed to the experimental pH for 24 h and blood was sampled. Urine flow rate, urine and plasma pH showed a significant trend to increase with the increase of water pH. Urinary Na⁺ excretion rate also increased and ammonia urinary excretion rate decreased with the increase of water pH. There was a significant trend to decrease volume, ammonia, Cl⁻ and Na⁺ urinary excretion rate with increasing mass in fish exposed to all pH levels studied. Plasma ammonia levels showed a slight decrease in fish exposed to water pH from 4·0 to 8·0, but those exposed to water pH 9·0 presented the highest ammonia levels. Most plasma ions and urinary excretion changes observed in silver catfish exposed to acidic or alkaline water were similar to those already detected in rainbow trout Oncorhynchus mykiss . In addition, the kidney and urinary bladder might participate on acid–base balance in silver catfish, since urine pH changed according to plasma pH.