[Visualization of local cortical defects in Charcot foot using microcomputed tomography]. / Visualisierung lokaler kortikaler Defekte im Charcot-Fuß mittels Mikrocomputertomographie.

BACKGROUND: In the pathogenesis of diabetic neuropathic osteoarthropathy (Charcot's foot) fractures cause chronic destruction of soft tissue and bone structure. To improve an early diagnosis of Charcot foot, modern diagnostic imaging is mainly based on magnetic resonance imaging (MRI), for example in relation to the detection of cortical bone fractures. OBJECTIVES: In this study we investigated the cortical microstructure in cases of Charcot foot with respect to fractures and porosity in order to visualize local cortical defects. This may substantiate recent efforts in a reclassification based on MRI. MATERIAL AND METHODS: Using microcomputed tomography (microCT) we investigated bone parameters, such as cortical thickness and porosity in order to quantify the local metatarsal microstructure in cases of Charcot foot. RESULTS: All bone samples showed a high degree of cortical porosity including pores that perforated the cortical bone. The data suggest that areas with reduced cortical thickness coincide with large cortical pores that may serve as initial points for fractures. Whether the detected microfractures are physiological or artefacts of preparation could not be determined. CONCLUSION: By means of microCT we were able to visualize and quantify the extent of cortical porosity for the first time in high resolution. The data suggest that both cortical fractures and cortical porosity play an important role in the pathogenesis in cases of Charcot foot.

Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase.

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.

Activation and inhibition of the calcium-release channel of isolated skeletal muscle heavy sarcoplasmic reticulum. Models of the calcium-release channel.

Calcium-independent calcium efflux from heavy sarcoplasmic reticulum (HSR) of skeletal muscle was found to be biphasic, with half-times of 2-6 s and 200-400 s for the first and second phase, respectively. Calcium-, AMP- and caffeine-induced calcium efflux was triphasic, with half-times of 0.05-0.2 s, 1-5 s and 100-400 s for the first, second and third phases, respectively. This very fast first phase is certainly due to calcium efflux via the calcium-release channel of HSR vesicles. Both ruthenium red and neomycin inhibited the first phase of the calcium-independent calcium efflux and the first phase of the calcium-, AMP- or caffeine-induced calcium efflux completely, whilst the second phase was fully inhibited by ruthenium red only and partially inhibited by neomycin at high concentrations, indicating that the second phase of calcium release also occurs via the calcium-release channel. Various models for calcium efflux through the release channel have been tested by simulation. Activation and inhibition of the channel-mediated calcium efflux from HSR cannot be explained by two states of the calcium-release channel (open or closed), but requires the existence of at least three states. A channel with one open state and two closed states, resulting in a rapid inactivation, is the most simple model compatible with the experimental data. According to this model, activation is assumed to reduce inactivation of the channel, whilst inhibition assumes an acceleration of channel inactivation. This mechanism most likely applies to neomycin. An additional open-blocked state has to be assumed for inhibition by ruthenium red.

[Calcium channel mediated calcium release from the rabbit skeletal muscle sarcoplasmic reticulum vesicle]. / Calciumfreisetzung über den Calciumkanal sarkoplasmatischer Retikulum-Vesikel des Kaninchenskelettmuskels.

Activation and inhibition of the calcium release channel of rabbit skeletal muscle heavy sarcoplasmic reticulum (HSR) was investigated by various methods. The calcium release channel is activated by binding of calcium in the micromolar range and by binding of adenine nucleotides in the millimolar range. Ruthenium red and neomycin are potent inhibitors of the channel at nanomolar to micromolar concentrations. Dantrolene inhibits the rate of caffeine-induced calcium release. Several models of the calcium release channel were considered to explain the three-phasic calcium release from HSR vesicles. Simulation of calcium efflux data according to various models suggest that the calcium release channel has at least three states. The experimental results can be explained by assuming one open and two closed states of the calcium release channel, but not by assuming one open and one closed state.

Inhibition of calcium release from skeletal muscle sarcoplasmic reticulum by calmodulin.

The effect of calmodulin on calcium release from heavy sarcoplasmic reticulum isolated from rabbit skeletal muscle was investigated with actively and passively calcium loaded sarcoplasmic reticulum vesicles and measured either spectrophotometrically with arsenazo III or by Millipore filtration technique. The transient calcium-, caffeine- and AMP-induced calcium release from actively loaded sarcoplasmic reticulum vesicles was reduced to 29%, 51% and 59% of the respective control value by 1 microM exogenous calmodulin. Stopped-flow measurements demonstrate that calmodulin reduces the apparent rate of caffeine-induced calcium release from actively loaded sarcoplasmic reticulum. The rate of calcium uptake measured in the presence of ruthenium red, which blocks the calcium release channel, was not affected by calmodulin or calmodulin-dependent phosphorylation of sarcoplasmic reticulum vesicles with ATP[S]. The rate of the calcium-, caffeine- and AMP-induced calcium release from passively loaded sarcoplasmic reticulum vesicles was reduced 1.4-2.0-fold by 1 microM exogenous calmodulin, i.e. the half-time of release was maximally increased by a factor of two, whilst calmodulin-dependent phosphorylation of a 57 kDa protein with ATP[S] had no effect. The data indicate that calmodulin itself regulates the calcium release channel of sarcoplasmic reticulum.

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump.

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.

Alteration of acylphosphate formation of cardiac sarcoplasmic reticulum ATPase by calmodulin-dependent phosphorylation.

The calcium-dependent acylphosphate formed by the calcium transport ATPase of cardiac sarcoplasmic reticulum and the calcium-, calmodulin-dependent phosphoester(s) of sarcoplasmic reticulum fractions formed by a calcium-, calmodulin-dependent membrane-bound protein kinase can be distinguished by removal of calcium and/or magnesium by EDTA or hydroxylamine treatment of the acid denaturated membranes. Both procedures decompose the acylphosphate with little effect on the phosphoester(s). Calmodulin-dependent phosphorylation (2.44 nmol/mg SR protein) reduces the apparent K(Ca) of the acylphosphate steady state level of the calcium transport ATPase from 0.56 to 0.34 microM free calcium, without affecting the maximum phosphoenzyme level (0.93 versus 0.89 nmol/mg protein), and has little, if any, effect on the Hill-coefficient (1.32 versus 1.54).

Correlation between calmodulin-dependent increase in the rate of calcium transport and calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum. Characterization of calmodulin-dependent phosphorylation.

The aim of the present study was to prove a correlation between the calmodulin-dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin-dependent phosphorylation. The dependence of phosphorylation on the total calmodulin concentration at 75 microM and 1 microM free calcium gave apparent calmodulin half-saturation constants Km (CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km (CaM) for the rate of calmodulin-stimulated calcium transport carried out at 1 microM calcium, but phosphorylated prior to the calcium uptake at 75 microM or 1 microM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of calcium transport and hydroxylamine-insensitive phosphoester formed by the calcium/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26-28-kDa or 9-11-kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37 degrees C and at 100 degrees C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. The data indicate that calmodulin-dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 microM and 0.61 microM in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 microM, 1.44 microM and 2.3 microM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum.

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.

Formation of magnesium-phosphoenzyme and magnesium-calcium-phosphoenzyme in the phosphorylation of adenosine triphosphatase by orthophosphate in sarcoplasmic reticulum. Models of a reaction sequence.

The aim of the present study was to test simple reaction sequences which describe calcium-independent plus calcium-dependent phosphorylation of sarcoplasmic reticulum transport. ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg . E . Pi), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium-phosphoenzyme (Mg . E-P). Phosphorylation, performed at pH 7.0 20 degrees C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent magnesium half-saturation constant and an increase in maximum phosphoprotein formation with increasing calcium loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent orthophosphate half-saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X-537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low-affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation. Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E . Mg . Pi or Mg . E-P or to both, but neither to E. Mg nor to E . Pi. Secondly, calcium-independent and calcium-dependent phosphoproteins are magnesium-phosphoenzymes. Calcium-dependent phosphoenzyme is a magnesium-calcium-enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg . E-P . (Cai)2], and not a 'calcium-phosphoprotein' without bound magnesium.

[Characterization of the calcium transport cycle of sarcoplasmic reticulum by inorganic phosphate including the function of magnesium (author's transl)]. / Uber die durch Orthophosphat charakterisierbare Reaktionssequenz des Calciumtransportzyklus sarkoplasmatischer Retikulummembranen unter Einbeziehung der Funktion des Magnesiums.

The present study presents experiments on ATP-Pi exchange and phosphorylation of the calcium-transport-ATPase of sarcoplasmic reticulum vesicles by orthophosphate under conditions of ATP-Pi exchange, as well as on Ca-independent and Ca-dependent phosphorylation in the absence of ATP, ADP and calcium outside. The rate of the ATP-Pi exchange correlates with the phosphoprotein steady state level labelled from orthophosphate. Ca-independent phosphorylation is due to magnesium-phosphoprotein formation and Ca-dependent phosphorylation is due to magnesium-calcium-phosphoprotein formation. A reaction sequence which probably accounts for phosphorylation of the transport enzyme by orthophosphate and its significance in characterizing the intermediate steps of the calcium transport cycle is discussed.

pH and temperature dependence of adenosine uptake in human erythrocytes.

Kinetic analysis of the saturable adenosine uptake in human erythrocytes suggests the existence of two saturable components, distinguished by different Km values (1.4 and 260 micron, respectively, at pH 7.4 and 25 degrees C). Both components were abolished by p-nitrobenzylthioguanosine or dipyridamole. Total uptake was significantly higher at pH 8 than at pH 7 at adenosine concentrations above 2 micron. The increase in uptake at the higher pH was brought about mainly by an increase in the maximum rate of transport of the low-affinity uptake system. With rising temperature the Km and the V of both uptake components increased. No transition temperature was observed between 12 and 37 degrees C.

The determination of the myocardial extracellular space in the cat in vivo: a comparative methodological study.

The myocardial extracellular space (ECS) was determined in cats in vivo by means of the ECS indicators, inulin and sulfanilic acid and, additionally, as glucose space. Experiments were carried out in cats subjected to bilateral kidney ligation (single injection) and in intact cats (single injection or infusion of indicator). The heart was clamped by instant deep-freezing in situ; this technique was compared, in renally-ligated cats, with excision and subsequent freezing of the heart in liquid nitrogen. The myocardial blood content was significantly decreased, and the myocardial lactate concentration significantly increased in excised, as compared with in situ-clamped hearts. Renally-ligated cats showed marked hypotension. The mean myocardial blood content was also significantly lower than in intact animals. A highly significant correlation was found between myocardial blood content and blood pressure for all experiments with instant deep-freezing. The mean inulin ECS value in renally-ligated cats was 22.7 +/- 1.5 ml. In accordance with the fact that the ECS is dependent on the tissure blood content, the corresponding values in intact animals were significantly higher, 25.9 +/- 2.5 ml (single injection) and 26.3 +/- 3.9 ml (infusion), calculated per 100 g tissue wet weight. Similar values were obtained for the glucose and sulfanilic acid ECS. If the interstitial space, an expression independent of the tissue blood content is used as space parameter, no significant differences were found under any of the present experimental conditions.