Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.     

The calcineurin inhibitor gossypol impairs growth, cell signalling and development in Dictyostelium discoideum

The Dictyostelium genome harbors single copy genes for both the catalytic and regulatory subunits of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since molecular genetic approaches to reduce the expression of these genes have failed so far, we attempted to pharmacologically target calcineurin activity in vivo by using the recently described calcineurin inhibitor, gossypol. Up-regulation of expression of the gene for the Ca2+-ATPase PAT1 in conditions of Ca2+ stress was reduced by gossypol. Dictyostelium wild-type cells treated with 12.5-100 microM gossypol showed reduced growth rates and impaired development. In addition, cell signalling was affected. A cell line that overproduces the catalytic subunit of calcineurin was more resistant to gossypol.     

Interaction of bovine rhodopsin with calcium ions

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10^–9 M (using EGTA or EDTA) to 15×10^–3 M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1).The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10^–7 M calcium (by adding EGTA), the other containing 10^–3 M calcium (Table 2).Abbreviations A absorption - A absorption change - CTAB N-Cetyl-N,N,N-trimethylammoniumbromide - E₇₀₀ extinction at =700 nm - EDTA ethylenediamine-NNNN-tetraacetic acid - EGTA 2,2-ethylenedioxybis [ethyliminodi (acetic acid)] - MI metarhodopsin I - MII metarhodopsin II - Rh rhodopsin - ROS rod outer segment This work is based upon a Ph. D. dissertation (Nöll, 1974) and was presented in part at the Jahrestagung der Deutschen Gesellschaft für Biophysik, Freiburg, Germany, October 1974     

Control of contractility in Spirostomum by dissociated calcium ions

The freshwater protozoan, Spirostomum ambiguum, exhibits generalized contraction when electrically stimulated with a DC pulse. Light and electron microscopic studies show a subcortical filamentous network, believed responsible for generating contractile tension, in association with vesicles which were shown to accumulate calcium oxalate precipitates. Organisms microinjected with the calcium-sensitive, bioluminescent protein, aequorin, emit light when stimulated to contract. Analyses of cinefilm records of electrically induced contraction indicate that contraction may occur up to 25 msec after the onset of stimulation at a point when the calcium-aequorin light emission is at a peak. The evidence shows that calcium release from an interval compartment is directly associated with the onset of contraction in Spirostomum, and that the removal of calcium, through some internal sequestering mechanism, signals relaxation.     

The in vivo modulation of dopamine synthesis by calcium ions: influences on the calcium independent release

To investigate the contribution of the dopamine (DA) synthesis to both the calcium-dependent and the carrier-mediated, mechanisms of DA release in the striatum, anaesthetized rats were locally superfused in the striatum with a push–pull cannula supplied with an artificial CSF containing tritiated tyrosine. DA, dihydroxyphenylacetic acid (DOPAC) and their respective specific activity were measured in effluent and used to evaluate changes in the DA synthesizing rate. Excluding calcium ions from the CSF only partially reduced spontaneous DA release (70%) still leaving a possible carrier-mediated DA release. This effect was not additive with a local superfusion with 0.1 mM a-methyl-p-tyrosine, a blocker of DA synthesis, suggesting that synthesis could already be reduced by calcium-free superfusion. Local superfusion with 100 μM cadmium in the presence or not of calcium ions, increased the DA release (220 and 350%, respectively), simultaneously reducing DA synthesis. Local application of 1 μM calcium ionophore (A23187) was without effect on the basal release of DA but enhanced DA synthesis and increased the amphetamine-evoked and carrier-mediated amine release. We conclude that DA synthesis can be a modulatory process of the firing-independent and carrier-mediated amine release while it weakly affects the classical calcium-dependent release.     

Activation of a pea membrane protein kinase by calcium ions

Membranes from the buds of Pisum sativum L. contain a protein kinase which is activated 5- to 15-fold by micromolar levels of calcium. Best calcium activations were found with light-membrane fractions, and on density gradients these band at a similar position to the plasma membrane. Other heavier membranes, however, also contain a calcium-dependent protein kinase. The activity of the calcium-dependent protein kinase is inhibited by added phospholipids and phospholipase, in contrast to protein-kinase C. Calcium-dependent protein-kinase activity can be inhibited by 40% by low concentrations of the calmodulin inhibitor, trifluoperazine, but inhibitions are detected only after prior incubation of the membranes for some hours in ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid. Substantial calcium-dependent protein-kinase activity remains uninhibited by trifluoperazine indicating that there may be calmodulin-dependent and calmodulin-independent, but calcium-activated, protein kinases in pea membranes. The calcium-activated protein kinase seems to be intrinsically bound to membranes and only slight or partial solubilisation is obtained by the detergents nonidet P-40, (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate or octyl glucose. Better solubilisation is obtained by acetone treatment. There is some retention of calcium activation after partial solubilisation. A calcium-independent protein kinase has also been detected in membrane preparations; it has a substrate specificity different from that the calcium-dependent enzyme. Our results indicate, therefore, that there may be at least three protein kinases attached to pea shoot membranes.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - TFP trifluoperazine     

Substructure of solitary cilia in mouse kidney

Summary Recent scanning electron microscopic studies confirm the presence of solitary cilia on most epithelial cells along the mammalian nephron and collecting ducts.By transmission electron microscopy we have found that the axonemata of such cilia consist of a maximal number of 9 doublet and no singlet filaments. 10% of the cross-sectioned cilia contain 9 doublets arranged in a peripheral ring (9+0 pattern). 30 % of the cross-sections contain 8 or 7 doublets in peripheral ring and 1 or 2 doublets in the central region (8+1 and 7+2 patterns). Serial sections and goniometer tilt reveal the central doublets to originate as dislodged peripheral doublets. 60% of the sectioned cilia contain filament numbers between 8 and 4. In patterns of 5 and 4 filaments single microtubules predominate.The functional significance of these atypical cilia is discussed.We are indebted to Prof. B. Afzelius and Prof. Th. Brun for valuable information and discussions during this work. The technical assistance of Miss K. Weltzin, Mr. E. Erichsen, Mr. R. Jensen and Mr. J. Røli is greatly appreciated     

Coupled calcium release channels and their regulation by luminal and cytosolic ions

Contraction in skeletal and cardiac muscle occurs when Ca²⁺ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca²⁺ release channels. Several isoforms of the RyR exist throughout the animal kingdom, which are modulated by ATP, Ca²⁺ and Mg²⁺ in the cytoplasm and by Ca²⁺ in the lumen of the SR. This review brings to light recent findings on their mechanisms of action in the mammalian isoforms RyR-1 and RyR-2 with an emphasis on RyR-1 from skeletal muscle. Cytoplasmic Mg²⁺ is a potent RyR antagonist that binds to two classes of cytoplasmic site, identified as low-affinity, non-specific inhibition sites and high-affinity Ca²⁺ activation sites (A-sites). Mg²⁺ inhibition at the A-sites is very sensitive to the cytoplasmic and luminal milieu. Cytoplasmic Ca²⁺, Mg²⁺ and monovalent cations compete for the A-sites. In isolated RyRs, luminal Ca²⁺ alters the Mg²⁺ affinity of the A-site by an allosteric mechanism mediated by luminal sites. However, in close-packed RyR arrays luminal Ca²⁺ can also compete with cytoplasmic ions for the A-site. Activation of RyRs by luminal Ca²⁺ has been attributed to either Ca²⁺ feedthrough to A-sites or to Ca²⁺ regulatory sites on the luminal side of the RyR. As yet there is no consensus on just how luminal Ca²⁺ alters RyR activation. Recent evidence indicates that both mechanisms operate and are likely to be important. Allosteric regulation of A-site Mg²⁺ affinity could trigger Ca²⁺ release, which is reinforced by Ca²⁺ feedthrough.     

Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation

To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus-like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast-expressed VP1 was self-assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non-reducing SDS–PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.     

The absorption of calcium ions from the ovine reticulo-rumen

Net Ca²⁺ and Mg²⁺ absorption rates were measured in vivo from buffer solutions placed in the washed reticulo-rumen, isolated in situ in 30 conscious, trained sheep. An increase in concentration of short chain fatty acids (SCFA) in the buffer, over the range 0–50 mM, was shown to stimulate the net rates of absorption of Ca²⁺ and Mg²⁺ ions from the rumen. Similarly, the results of in vitro experiments, carried out with ovine rumen epithelium mounted in short-circuited Ussing chambers, showed that the absence of SCFA from the chamber fluid resulted in a reduction in J_net Ca²⁺ caused by reduced flux of Ca²⁺ ions in the mucosal to serosal direction (J_ms Ca²⁺). The addition of 1 mM acetazolamide, an inhibitor of carbonic anhydrase, to the ruminal buffer used in the in vivo experiments led to significant reductions in the net absorption rates of Ca²⁺and Mg²⁺ ions in the presence of SCFA (50 mmol l⁻¹) but not in the absence of SCFA. However, in the in vitro experiments, the addition of 60 μM ethoxyzolamide had no significant effect on J_net Ca²⁺. A reduction in pH of the intraruminal buffer in vivo from 6.8 to 5.4 led to significant increases in the net absorption rates of Ca²⁺and Mg²⁺ ions, an effect which was duplicated for Ca²⁺ in preliminary in vitro experiments in which the pH of the mucosal buffer was reduced from 7.4 to 5.4. This stimulatory effect was confined to J_ms Ca²⁺ and J_net Ca²⁺. Ussing chambers were also used to demonstrate that J_net Ca²⁺ was reduced by a high transmural potential difference (PD), caused by voltage clamping, independently of the mucosal K⁺ concentration. Both unidirectional Ca²⁺ fluxes consisted of a PD-dependent and a K⁺-insensitive PD-independent component. The latter may be represented by a Ca²⁺/2H⁺ antiporter. It is postulated that SCFA, and to a lesser extent H₂CO₃, can stimulate J_ms Ca²⁺ by activation of an apical Ca²⁺/2H⁺ antiporter through the provision of protons within the ruminal epithelial cell. A mild reduction in ruminal pH may also lead to a similar stimulation of this putative electroneutral exchange. Accepted: 26 July 2000     

FGF is essential for both condensation and mesenchymal-epithelial transition stages of pronephric kidney tubule development

The pronephros is a transient embryonic kidney that is essential for the survival of aquatic larvae. It is also absolutely critical for adult kidney development, as the pronephric derivative the wolffian duct forms the ductal system of the adult kidney and also triggers the condensation of metanephric mesenchyme into the adult nephrons. While exploring Xenopus pronephric patterning, we observed that epidermally delivered hedgehog completely suppresses pronephric kidney tubule development but does not effect development of the pronephric glomus, the equivalent of the mammalian glomerulus or corpuscle. This effect is not mediated by apoptosis. Microarray analysis of microdissected primordia identified FGF8 as one of the potential mediators of hedgehog action. Further investigation demonstrated that SU5402-sensitive FGF signaling plays a critical role in the very earliest stages of pronephric tubule development. Modulation of FGF8 activity using a morpholino has a later effect that blocks condensation of pronephric mesenchyme into the pronephric tubule. Together, these data show that FGF signaling plays a critical role at two stages of embryonic kidney development, one in the condensation of the pronephric primordium from the intermediate mesoderm and a second in the later epithelialization of this mesenchyme into the pronephric nephron. The data also show that in Xenopus, development of the glomus/glomerulus can be uncoupled from nephron formation via ectopic hedgehog expression and provides an experimental avenue for investigating glomerulogenesis in the complete absence of tubules.     

Characterization of mapacalcine-sensitive Ca(2+) channels in rat kidney

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.     

Differential effect of calcium ions on the cytosolic and mitochondrial thioredoxin reductase

The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.     

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions

Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.     

Exchange characteristics of calcium ions bound to anthrax protective antigen

Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1(') (amino acid residues 168-258). We showed that these ions are stably bound and exchange with free 45Ca(2+) only slowly (t(1/2) approximately 4.0 h). Dissociation is the rate-limiting step. PA(63), the heptameric prepore form of PA, showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca(2+) within PA plays primarily a structural role, maintaining domain 1(') in a conformation that allows PA(63) to oligomerize and bind the enzymatic moieties of the toxin.     

Insights into kidney stem cell development and regeneration using zebrafish

Kidney disease is an escalating global health problem,for which the formulation of therapeutic approaches using stem cells has received increasing research attention.The complexity of kidney anatomy and function,which includes the diversity of renal cell types,poses formidable challenges in the identification of methods to generate replacement structures.Recent work using the zebrafish has revealed their high capacity to regenerate the integral working units of the kidney,known as nephrons,following acute injury.Here,we discuss these findings and explore the ways that zebrafish can be further utilized to gain a deeper molecular appreciation of renal stem cell biology,which may uncover important clues for regenerative medicine.