Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle's loop

A calcium-sensing receptor (CaR) has functionally been described in the cortical thick ascending limb of Henle's loop (CTAL) of rat and mouse. This G protein-coupled receptor activates phospholipase C and increases the intracellular Ca2+ concentration. We observed that in the mouse CTAL cAMP formation, induced by 10(-8) mol/l AVP, was inhibited by more than 90% when the extracellular Ca2+ concentration ([Ca2+]e) was increased from 0.5 to 3 mmol/l. Measurements of transepithelial potential difference (PDte) in rat and mouse CTAL and medullary thick ascending limb (mTAL) segments and of transepithelial ion net fluxes in the mouse CTAL (isotonic perfusion conditions: 150 mmol/l NaCl in the lumen and bath) showed that an increase in the [Ca2+]e had no effect on basal and arginine vasopressin (AVP, 10(-10) mol/l)-stimulated transepithelial PDte, NaCl and Mg2+ transport. However, Ca2+ reabsorption was strongly inhibited by increased [Ca2+]e. Addition of AVP reversed this inhibitory effect of increased [Ca2+]e. Under hypotonic perfusion conditions (lumen 50 mmol/l NaCl; bath 150 mmol/l NaCl), a high [Ca2+]e induced a 50% decrease in Mg2+ reabsorption which was restored by AVP. Under these conditions, the effects on Ca2+ transport described above were still observed. In conclusion, activation of the CaR in the mouse TAL has no effect on basal and AVP-stimulated transepithelial NaCl reabsorption despite its large inhibitory effect on cAMP synthesis. The CaR, however, could play a role in the regulation of transepithelial Ca2+ and Mg2+ reabsorption.     

Role of calcium in exocrine pancreatic secreation. VI. Characteristics of the paracellular pathway for divalent cations

(1) The transepithelial permeability for Ca2+ and Mg2+ in the isolated rabbit pancreas has been studied. (2) Values for the permeability of the unstimulated pancreas were obtained either by adding radioactive tracers to the bathing medium and measuring their concentration in the secreted fluid under steady-state conditions, or by analysis of the Ca2+ and Mg2+ concentrations in the secreted fluid after correction for protein-bound divalent cations. (3) Both methods give almost the same results: 27 and 26% for Ca2+ and 21 and 18% for Mg2+, respectively; both values being expressed as the percentage of the concentrations in the bathing medium. (4) The amounts of Ca2+ and Mg2+, appearing in the secretory fluid after correction for protein-bound cations, are linearly related to the extracellular Ca2+ and Mg2+ concentrations in the bathing medium, which indicates passive permeation. The two cations appear to pass through the paracellular route in their hydrated form. (5) Stimulation with carbachol or pancreozymin causes an increase in the paracellular permeability. This increase is approximately equal for the two divalent cations. Its time dependence and magnitude depend on the concentration of the stimulant rather than on the type of stimulant.     

Role of calcium in exocrine pancreatic secretion. III. Comparison of calcium and magnesium movements in rabbit pancreas

1. Calcium and magnesium movements in the isolated rabbit pancreas and in rabbit pancreas fragments are compared in a qualitative and quantitative way. 2. At the basal secretion rate calcium and magnesium are present in the secreted fluid in concentrations of about 30% of their concentrations in the bathing medium. 3. Addition of 10(-6) M carbachol to the bathing medium results in enzyme secretion accompanied by calcium and magnesium release, in divalent cation-free medium as well as in a complete medium. 4. The secretion of each divalent cation is the sum of two components: an extracellular flux and a flux of protein-associated cations, the so-called secretory flux. 5. The extracellular flux is proportional to the concentration of the divalent cation in the bathing medium. The secretory flux is not dependent on the presence of the divalent cation in the bathing medium, but is proportional to the amount of protein secreted. About 25 nmol of each cation is secreted per mg protein. 6. Ca2+ and Mg2+ can be nearly completely separated from the digestive enzymes by gel filtration. They equilibrate completely with their radioactive isotopes added to the sample just before elution, indicating that the cations are rapidly exchangeable after secretion. 7. Efflux studies on rabbit pancreas fragments, pre-loaded with 45Ca2+, show a carbachol-stimulated 45Ca2+ efflux (the stimulatory flux) in addition to a release of amylase. Fragments pre-loaded with 28Mg2+ do not show carbachol stimulation of the tracer efflux. 8. These studies indicate that calcium and magnesium behave quite similarly with respect to the extracellular and secretory fluxes. The absence of a stimulatory flux for magnesium, suggests that the increase of the cytoplasmic calcium concentration plays a specific role in the stimulus-secretion coupling of pancreatic enzyme secretion.     

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In order to study fluxes of phosphate (Pi), Ca and Mg into the rat proximal tubule, a modification of the split-droplet microinjection technique was used. Injected fluids were isotonic solutions containing no Pi, Ca or Mg. The initial NaCl concentration of the injectates was either (a) 115 mM(l (which resulted in net fluid entry into the lumen), (b) 125 mM/l (no net fluid movement) or (c) 150 mM/l (net reabsorption of fluid). Injected droplets were subsequently collected from the nephron and their ionic concentrations determined using electron probe analysis. All 3 ions entered the tubular lumen. For the 115 mM and 125 mM NaCl injectates, Pi concentration increased for the first 15 s, then reached steady values of 2.07 mM/l and 2.30 mM/l respectively. Using 150 mM NaCl as injectate, Pi concentration increased for only 10 s, and then reached an average value of 2.04 mM/l. Ca and Mg concentrations in reaspirated droplets showed no correlation with time, indicating that entry into the lumen was almost immediate. The mean Ca concentration using 115 mM NaCl injectate was 1.63 mM/l, higher than with equilibrated or reabsorbed injectates (1.01 and 1.15 mM/l respectively). Mg concentration following injection of 115 mM NaCl solution (0.45 mM/l) was lower than with the other 2 injectates (0.92 and 0.85 mM/l). It is suggested that Pi and Mg enter the proximal tubular lumen from the tubular cells, while Ca entry may be transtubular and take place via intercellular pathways.     

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked Xenopus laevis embryos

Uptakes of (28)Mg at 10 s were measured at 0.1 and 1mM MgCl(2), to mainly represent one or other of the two uptake mechanisms earlier shown to be present in rat jejunal brush border membrane vesicles, one with an apparent KT of 0.2 mM, the other in the millimolar range. Both mechanisms had an optimal temperature close to 28 degrees C, inactivation at 37 degrees C being more acute for the low affinity mechanism (55 percent, P < 0.01). Both mechanisms were equally stimulated by an electrical potential difference (negative inside the vesicles) imposed by a potassium gradient and not affected by the nature of the anion accompanying magnesium. At 0.1 mM MgCl(2), the uptake was increased by an outwardly directed proton gradient, pH 8.2 outside and 7.4 inside (38 percent, P < 0.05), but not depressed when the gradient was in the opposite direction, pH 6.6 outside and 7.4 inside. It was trans-stimulated by magnesium, strongly inhibited by amiloride and to a smaller extent by furosemide, but uninfluenced by 0.1 mM NaCl or by 100 mM NaCl, NaSCN or KCl. A slight but significant inhibition (20-30 per cent) was recorded in the presence of 0.1 mM CoCl(2), NlCl(2) or BaCl(2). At 1 mM MgCl(2), the uptake was not influenced by pH gradient, was not trans-stimulated by Mg and was not affected by furosemide. A 40 percent inhibition by amiloride was, however, recorded. Also 100 mM NaCl or KCl doubled the uptake, while 1 mM NaCl or 100 mM NaSCN did not affect it. In contrast, all the divalent cations tested produced an inhibition (from 60 to 12 percent) in the following order: Co > or = Mn > Ca > or = Ni> Ba > Sr, when used at the same concentration as magnesium. The results showed that cobalt and calcium were not true competitors. In conclusion, two distinct mechanisms would operate magnesium entry at the brush border: (1) an electrogenic high affinity Mg/Mg,H exchange, sensitive to amiloride and furosemide, and (2) an electrogenic low affinity mechanism, inhibited by the presence of several divalent cations and dependent on the presence or activity of alkaline phosphatase.     

Binding of ADP to sarcoplasmic reticulum Ca(2+)-ATPase in the absence of Mg2+ is specifically inhibited by thapsigargin: observations on the ligand stoichiometry

The conditions of nucleotide binding to native, though partly purified, Ca(2+)-ATPase from SR as well as the stoichiometry of nucleotide and strontium binding and the phosphorylation capacity was reevaluated. Binding of MgADP appeared to be aberrant whereas even high-affinity binding of [14C]-ADP took place in the absence of Mg2+. Also low-affinity ATP binding was possible in the absence of divalent cations. A heterogeneity in ADP binding compatible with a two-component model in the absence of thapsigargin was changed to an apparent homogeneity of low-affinity receptors following a mole:mole interaction of enzyme and thapsigargin. Since the affinity of both components was reduced by thapsigargin, high- as well as low-affinity ADP binding seem to be specific and probably to the substrate receptor proper. Analysis of ADP binding isotherms in the absence of Mg2+ according to a model of two independent populations of sites was compatible with a binding capacity of 8.49 +/- 0.43 nmoles/mg protein corresponding to a molecular mass of 118 +/- 6 kD per ADP site. The same total binding capacity was found for ATP. The phosphorylation capacity corresponded to more than one and less than two approximately P per two 110-kD peptides (formally one approximately P per 154 kD protein). Specific binding of Ca2+ and the congener Sr2+ to SR Ca(2+)-ATPase was compatible with their interaction with a single population of sites. The binding capacity was equal to one divalent cation per nucleotide binding peptide. The binding of one nucleotide and one divalent cation per approximately 110 kD peptide and the absence of cooperativity in divalent cation binding might imply that Ca(2+)-ATPase works as a monomer.     

Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium

One of the most important factors controlling blood pressure is the total body Na+ content, which depends upon Na+ intake and excretion. The kidney influences body Na+ content by regulating the tubular absorption of the Na+ filtered through the glomeruli. Thus, the regulation of Na+ absorption in the tubules of the kidney plays an important role in controlling blood pressure. More than 99% of the Na+ passing through the glomerulus is reabsorbed in the kidney. About 90% of the filtered Na+ through the glomerulus is reabsorbed in the proximal tubule and the ascending limb of the loop of Henle. The remainder of the Na+ absorption occurs in the distal nephron. This process is regulated by hormones such as aldosterone and antidiuretic hormone (ADH), and also by the osmolality of the plasma. These observations suggest that the regulation of Na+ transport in the distal nephron by hormones and osmolality plays an important role in the control of blood pressure. The distal nephron is composed of two different types of epithelial cells: the principal cell and the intercalated cell. The latter is also composed of two types of cells: alpha and beta intercalated cells. In addition to Na+ absorption, the distal-nephron epithelial cells also participate in K+ and H+ secretion. Na+ absorption is mediated through the principal cell, which also contributes to K+ secretion, whereas H+ is secreted through both types of intercalated cells, alpha and beta, in different ways. There are, in general, two steps in the transepithelial ion movement across the epithelium, including the distal-nephron epithelium. For example, in the case of Na+ absorption, one is the entry step of Na+ across the apical membrane and the other is the extrusion step of Na+ across the basolateral membrane. This means that there are two major regulatory sites of transepithelial Na+ absorption: namely, regulation of the entry and extrusion steps of Na+. It is generally thought that the entry step of Na+ across the apical membrane is the rate-limiting step in the transepithelial Na+ transport and that Na+ channels in the apical membrane play an important role as an entry step of Na+ and are regulated by hormones and plasma osmolality. In this review, we describe the regulatory mechanisms of Na+ absorption in renal distal-nephron epithelium by aldosterone, ADH and osmolality. Further, we will review the regulatory mechanisms of Cl- transport, which also plays an important role in Na+ transport as a major counter ion, and discuss other roles of Cl- in the active regulation of Na+ transport.     

Influence of extracellular fluid volume expansion on magnesium, calcium and phosphate handling along the rat nephron

Renal tubular handling of P, Ca, Mg and Na was studied in the rat both before and during mild hypertonic NaCl loading (ECVE), using micropuncture and clearance techniques and electron microprobe analysis. Micropuncture was performed at the late proximal and early distal tubule sites. ECVE significantly increased the urinary output of all four elements. In the case of Mg, the increase was relatively small and depended on slight but statistically unsignificant inhibition of reabsorption all along the entire length of the nephron. For Ca, it depended on the inhibition of proximal reabsorption, partially compensated by increased reabsorption along the loop. For P, it depended on proximal inhibition, no important net phosphate movement occurring in the loop during both periods. Ca reabsorption was highly correlated to that of sodium along the proximal tubule and Henel's loop, Ca and Mg reabsorption were closely related to the load delivered at the beginning of the structure. These observations are compatible with the view that tubular reabsorption of Ca and Mg is concentration rather than Tm limited, and that reabsorption of Ca, unlike that of Mg, is linked to the movements of sodium. Following ECVE, the difference between early distal and urinary deliveries increased significantly for Ca and P, but not for Mg. For phosphate, this difference accounted for by 45% of the delivery at the early distal tubule site, at variance with microinjection data obtained in the rat under similar salt loading conditions, which indicated that 17% only of the phosphate distal delivery were reabsorbed along the terminal segments. This discrepancy is discussed in terms of nephron functional heterogeneity.     

Oligomerization and divalent ion binding properties of the S100P protein: a Ca2+/Mg2+-switch model

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2+-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2+, Mg2+ and Zn2+) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-filtration chromatography, circular dichroism and fluorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2+-binding sites in the S100P molecule with different affinity; the high affinity binding site has an apparent binding constant of approximately 10(7) M-1 and the low affinity binding site has an apparent binding constant of approximately 10(4) M-1. (2) The high and low affinity Ca2+-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2+ (with relatively low affinity approximately 10(3) M-1), but not Mg2+. The N-terminal site binds Mg2+ with the apparent binding constant approximately 10(2) M-1. (4) Binding of Ca2+ to the C-terminal site and binding of Mg2+ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2+ and millimolar for Mg2+). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2+. On the basis of these observations a plausible model for S100P as a Ca2+/Mg2+ switch has been proposed.     

Formation of giant liposomes promoted by divalent cations: critical role of electrostatic repulsion

Spontaneous formation of giant unilamellar liposomes in a gentle hydration process, as well as the adhesion energy between liposomal membranes, has been found to be dependent on the concentration of divalent alkali cations, Ca2+ or Mg2+, in the medium. With electrically neutral phosphatidylcholine (PC), Ca2+ or Mg2+ at 1-30 mM greatly promoted liposome formation compared to low yields in nonelectrolyte or potassium chloride solutions. When negatively charged phosphatidylglycerol (PG) was mixed at 10%, the yield was high in nonelectrolytes but liposomes did not form at 3-10 mM CaCl2. In the adhesion test with micropipette manipulation, liposomal membranes adhered to each other only in a certain range of CaCl2 concentrations, which agreed with the range where liposome did not form. The adhesion range shifted to higher Ca2+ concentrations as the amount of PG was increased. These results indicate that the divalent cations bind to and add positive charges to the lipids, and that membranes are separated and stabilized in the form of unilamellar liposomes when net charges on the membranes produce large enough electrostatic repulsion. Under the assumption that the maximum of adhesion energy within an adhesive range corresponds to exact charge neutralization by added Ca2+, association constants of PC and PG for Ca2+ were estimated at 7.3 M(-1) and 86 M(-1), respectively, in good agreement with literature values.     

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.     

Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.     

Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced CL reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.     

A Ba(2+)-insensitive K+ conductance in the basolateral membrane of rabbit cortical thick ascending limb cells

The nature of the K+ exit across the basolateral membrane of microperfused rabbit cortical thick ascending limbs (cTALs) was investigated using the transepithelial and transmembrane potential difference (PDte, PDbl) and conductance measurements. An increase in bath K+ concentration from 4 to 10, 25, 50 mmol/l depolarized the basolateral membrane in a concentration-dependent manner, accompanied by a decrease in the fractional resistance of the basolateral membrane (FRbl). The Cl- channel blocker, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), did not prevent these effects. The effect of Ba2+ on PDbl was bimodally distributed: paradoxically, in the tubules in which Ba2+ largely depolarized, the effects on PDbl of the bath K+ concentration increases were not inhibited by extracellular Ba2+, in tubules in which Ba2+ moderately depolarized, Ba2+ partially inhibited the K+ concentration increase-induced depolarization of the basolateral membrane. However, the parallel decrease in FRbl was Ba2+ insensitive, indicating that the K+ channel of the basolateral membrane was not modified by extracellular Ba2+. The Ba(2+)-induced depolarizations were prevented by furosemide suggesting that Ba2+ acts by inhibiting basolateral KCl extrusion. Finally, the K+ concentration increase-induced depolarizations were insensitive to tetraethylammonium, charybdotoxin, apamin and verapamil. In conclusion, the present study provides evidence that, in addition to a Ba(2+)-sensitive KCl cotransport system, the basolateral membrane of rabbit cTAL cells possesses a K+ conductance which is insensitive to extracellular Ba2+.     

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The regulatory properties of the divalent metal ions Mg2+, Ca2+ and Mn2+ on the activity and kinetic behaviour of rat liver microsomal cholesterol esterase were studied in vitro. Mg2+ and Ca2+ exhibited similar concentration and preincubation time-dependent increases in esterase activity, with maximal stimulation at a concentration of 2 mM. However, Mn2+ had no effect at this concentration but displayed a potent inhibitory effect at concentrations above 20 mM. Activation of cholesterol esterase by Mg2+ and Ca2+ was selective in relation to i) the changes that cations produced in the enzyme kinetic constants, and ii) the chelating agents that reversed the metal ion-induced activation. Hence, the maximum rate of cholesterol ester hydrolysis doubled in the presence of Mg2+ and activation was reversed by EDTA, whereas a significant decrease in the apparent Km for cholesterol oleate was found when Ca2+ was added and this effect was blocked by ATP and EGTA. Both cations were able to reactivate cholesterol ester hydrolase activity in metal-depleted microsomes.     

Mechanism of fluid secretion common to aglomerular and glomerular kidneys

Isolated renal proximal tubules of sea water fish net secrete fluid in vitro. The principal electrolytes in secreted fluid are Na, Cl, Mg and S. Transepithelial voltages may be lumen-negative or -positive by a few millivolts, and transepithelial resistances are low partly due to high paracellular Na and Cl permeabilities. Transepithelial electrochemical potentials indicate secretion of Mg into the tubule lumen by active transport. As Mg concentration in secreted fluid rises, Na concentration falls. Surprisingly, these observations of fluid secretion are made in glomerular and aglomerular proximal tubules, suggesting a fundamental mechanism common to both. Central to this commonality appears to be their behavior as open Donnan systems. Mg actively secreted into the tubule lumen from which it cannot diffuse back into the peritubular medium causes the transepithelial secretion of diffusible Na and Cl. Water follows by osmosis. Since there is flow out of the distal end of the tubule Donnan equilibrium is not attained. Instead, a dynamic Donnan system is maintained, driven by active transport of Mg. A mathematical model of tubular electrolyte and fluid secretion confirms the operation of this open, dynamic Donnan system in aglomerular and glomerular proximal tubules.     

High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium

The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 mM) in normal Ca2+ medium. The inhibitory effects of high Mg2+ became apparent concomitantly with an increase in [Mg2+]i from 0.7 to 1.3 mM. High Mg2+ significantly inhibited TRH-induced PRL secretion in a dose-dependent manner in normal Ca2+ medium. TRH-stimulated inositol triphosphate (IP3) production was rather augmented by the replacement with high Mg2+ medium. In summary, high Mg2+ inhibits Ca2+ influx stimulated by TRH in the rat pituitary lactotropes, possibly with the involvement of [Mg2+]i increase. These results have general importance in relation to high Mg(2+)-induced suppression of the biological functions of cells.     

Multiple binding sites in the interaction between an extracellular fibrinogen-binding protein from Staphylococcus aureus and fibrinogen

Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the alpha-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.     

Low Na+ diet inhibits Na+ and water transport response to vasopressin in rat cortical collecting duct

BACKGROUND: We previously demonstrated that vasopressin (AVP) produces a sustained increase in Na+ reabsorption by the isolated perfused cortical collecting duct (CCD) from rats on a normal diet, and that this effect is synergistic with that of pharmacological doses of deoxycorticosterone (DOC) or physiological levels of aldosterone. The present experiments examined the effect of AVP under the more physiological circumstances when plasma aldosterone was elevated by prior volume depletion. METHODS: Rats were volume depleted by a single dose of furosemide followed by a low-salt diet (0.3% NaCl) for four to nine days. Some of these rats were also implanted with a pellet containing 2.5 mg DOC. Rats in a third group were not injected with furosemide but were implanted with the DOC pellet and maintained on a standard (approximately 1% NaCl) diet. CCD were perfused and the lumen-to-bath Na+ flux (JNA), transepithelial voltage (VT), and osmotic water permeability (Pf) were measured in the presence and absence of 200 pm AVP. RESULTS: Although Na+ depletion by a single injection of furosemide and the low salt diet elevated plasma aldosterone and Vt, JNA remained low and there was a decreased response to AVP in comparison with DOC-treated rats on a standard diet. In CCD from rats on the low salt-diet with DOC, JNa was less than observed in CCD from DOC-treated rats on a standard diet. AVP-dependent Pf in CCD from rats on the low salt-diet, with or without DOC treatment, was also markedly lower. CONCLUSIONS: We interpret the results to demonstrate that maximal rates of Na+ reabsorption in the CCD depend not only on the synergistic stimulatory effects of aldosterone and AVP, but also require normal to high rates of salt delivery in vivo for the effects of the hormones on Na+ transport to be maximized in vitro.