Presence Of High Ca Research Articles

Single-channel biophysical properties of the left ventricular mitochondrial calcium uniporter complex

Mitochondrial calcium uniporter (MCU) forms a macromolecular complex composed by MCU, MICU1, MICU2, EMRE and MCUb, which confers its tissue specificity. However, the native single-channel properties and its regulation by kaempferol, a polyphenol known to enhance mitochondrial Ca 2+ uptake has never been investigated in left ventricles. Here, we aimed to characterize the single-channel properties of mouse left ventricular MCU complex incorporated in planar lipid bilayer (PLB) and its regulation by kaempferol. MCU was purified from mouse left ventricle isolated mitochondria, treated with proteinase K and digitonin to disrupt the outer mitochondrial membrane. Submitochondrial particles were subjected to BN-Page to verify the macromolecular complex integrity. MCU was then incorporated in PLB for electrophysiological recording. Voltage steps were applied from −60 mV to +60 mV and Ca 2+ concentration was increased from 1 μM to 1 mM at −40 mV. Currents were recorded in presence of kaempferol (10 μM) and ruthenium red was used to inhibit the single-channel activity. At 1 μM Ca 2+ , MCU displayed 4 opening states with a single-channel open probability (Po) of 0.10 ± 0.05 and a single-channel conductance of 0.07 ± 0.01 nS. In presence of kaempferol, MCU Po and conductance were slightly increased (0.18 ± 0.08 and 0.10 ± 0.02 nS, respectively) and the number of opening states remained unchanged. At 100 μM Ca 2+ , the Po (0.06 ± 0.004) and conductance (0.05 ± 0.01) were not significantly different from those with low Ca 2+ condition whereas the single-channel current displayed only the first two opening states. Kaempferol increased both the conductance (0.11 ± 0.02 nS) and Po (0.27 ± 0.10) and maintained the MCU in 4 opening states. To conclude, we recorded for the first time MCU single-channel isolated from mouse ventricle and demonstrated that Ca 2+ impacts the channel opening states while kaempferol increases Po and conductance in presence of high Ca 2+ .

P1412CARDIO PULMONARY AND CKD-MBD INTERACTIONS IN HEMODIALYSIS PATIENTS

Abstract Background and Aims Emerging evidence suggests that pulmonary hypertension (PHT) is closely associated with cardiac, pulmonary, and mineral bone diseases in hemodialysis patients. Previous results indicated that extra osseous calcifications might be a risk factor for PHT although results in human studies about this association have been conflicting. Multiple factors including abnormal calcium phosphate levels, uremia and inflammation are known to be important in vascular calcification development. In hemodialysis patients’ vascular calcifications may be evaluated by different techniques. The utilization of plain X-ray for screening vascular calcifications has been suggested by KDOQI and KDIGO .In this study, we examined the association between PHT, peripheral vascular calcification by plain X-Ray score, and echocardiographic parameters in hemodialysis patients. Method Chronic (> 6 months) hemodialysis patients (150) on a three times per week dialysis schedule were studied. Two-dimensional trans thoracic echocardiography was performed before dialysis. Global systolic function was evaluated by left ventricular ejection fraction (LVEF). Systolic pulmonary arterial pressure (SPAP) was recorded. Pulmonary hypertension was defined as, pulmonary artery pressure (PAP) greater than 35 mm Hg based on European Society of Cardiology Guideline. Vascular calcification score is evaluated in plain X-ray of pelvis and hands. Each film is divided into four sections. Any vascular calcification either in an irregular or linear pattern is considered. The presence of vascular calcifications in each section is rated as 1 and its absence as 0. Final score is the sum of all sections and ranges from 0 to 8.Demographic data and lab results including Calcium (Ca), Phosphorous (P), Ca X P product, 25OH-vitD and iPTH were collected for all enrolled patients. The effects of different peripheral vascular calcification scores in plain X-Ray, structural heart abnormalities in echocardiography and Ca-P-iPTH- vit D axis disorders on the development of pulmonary hypertension were evaluated to elucidate cardio, pulmonary and CKD-MBD interactions. Results Hemodialysis patients had a high prevalence of PHT (30%), which warrants special attention to this important complication in hemodialysis population. Severe peripheral VCs (VCs >3) were independent risk factor for PHT. In addition to the old age, duration of hemodialysis, and mitral valve disease, the presence of higher Ca- P product was another independent predictor of PHT in chronic hemodialysis patients. Conclusion Prevalence of PHT in dialysis patients seems to be high, and is closely associated with peripheral VC, mineral bone disorders and structural heart abnormalities suggesting further a strong cardio pulmonary renal interaction.

SAT-234 CARDIO-PULMUNARY AND CKD-MBD INTERACTIONS IN HEMODIALYSIS PATIENTS

Emerging evidence suggests that pulmonary hypertension (PHT) is closely associated with cardiac, pulmonary, and mineral bone diseases in hemodialysis patients. Previous results indicated that extra osseous calcifications might be a risk factor for PHT although results in human studies about this association have been conflicting. Multiple factors including abnormal calcium phosphate levels, uremia and inflammation are known to be important in vascular calcification development. In hemodialysis patients’ vascular calcifications may be evaluated by different techniques. The utilization of plain X-ray for screening vascular calcifications has been suggested by KDOQI and KDIGO. In this study, we examined the association between PHT, peripheral vascular calcification by plain X-Ray score, and echocardiographic parameters in hemodialysis patients. Chronic (> 6 months) hemodialysis patients (150) on a three times per week dialysis schedule were studied. Two-dimensional trans thoracic echocardiography was performed before dialysis. Global systolic function was evaluated by left ventricular ejection fraction (LVEF). Systolic pulmonary arterial pressure (SPAP) was recorded. Pulmonary hypertension was defined as, pulmonary artery pressure (PAP) greater than 35 mm Hg based on European Society of Cardiology Guideline. Vascularcalcification score is evaluated in plain X-ray of pelvis and hands. Each film is divided into four sections. Any vascular calcification either in an irregular or linear pattern is considered. The presence of vascular calcifications in each section is rated as 1 and its absence as 0. Final score is the sum of all sections and ranges from 0 to 8. Demographic data and lab results including Calcium (Ca), Phosphorous (P), Ca X P product, 25OH-vitD and iPTH were collected for all enrolled patients. The effects of different peripheral vascular calcification scores in plain X-Ray, structural heart abnormalities in echocardiography and Ca-P-iPTH- vit D axis disorders on the development of pulmonary hypertension were evaluated to elucidate cardio, pulmonary and CKD-MBD interactions. Hemodialysis patients had a high prevalence of PHT (30%), which warrants special attention to this important complication in hemodialysis population. Severe peripheral VCs (VCs >3) were independent risk factor for PHT. In addition to the old age, duration of hemodialysis, and mitral valve disease, the presence of higher Ca- P product was another independent predictor of PHT in chronic hemodialysis patients. View Large Image Figure ViewerDownload Hi-res image Download (PPT) Prevalence of PHT in dialysis patients seems to be high, and is closely associated with peripheral VC, mineral bone disorders and structural heart abnormalities suggesting further a strong cardio pulmonary renal interaction.

Sarcolipin Regulation of SERCA is Distinct from Phospholamban

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Our recent data suggest that SLN plays a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN not PLB to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. We used chemical cross-linking to study the dynamic protein-protein interaction. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca2+ causes uncoupling of the SERCA pump and increased heat production.

Intermittent hypoxia protects cerebral mitochondrial function from calcium overload

Hypoxia leads to Ca(2+) overload and results in mitochondrial uncoupling, decreased ATP synthesis, and neuronal death. Inhibition of mitochondrial Ca(2+) overload protects mitochondrial function after hypoxia. The present study was aimed to investigate the effect of intermittent hypoxia on mitochondrial function and mitochondrial tolerance to Ca(2+) overload. Wistar rats were divided into control and intermittent hypoxia (IH) groups. The IH group was subject to hypoxia for 4 h daily in a hypobaric cabin (5,000 m) for 7 days. Brain mitochondria were isolated on day 7 following hypoxia. The baseline mitochondrial functions, such as ST3, ST4, and respiratory control ratio (RCR = ST3/ST4), were measured using a Clark-type oxygen electrode. Mitochondrial adenine nucleotide concentrations were measured by HPLC. Mitochondrial membrane potential was determined by measuring rhodamine 123 (Rh-123) fluorescence in the absence and presence of high Ca(2+) concentration (0.1 M), which simulates Ca(2+) overload. Our results revealed that IH did not affect mitochondrial respiratory functions, but led to a reduction in AMP and an increase in ADP concentrations in mitochondria. Both control and IH groups demonstrated decreased mitochondrial membrane potential in the presence of high Ca(2+) (0.1 M), while the IH group showed a relative higher mitochondrial membrane potential. These results indicated that the neuroprotective effect of intermittent hypoxia was resulted partly from preserving mitochondrial membrane potential, and increasing mitochondrial tolerance to high calcium levels. The increased ADP and decreased AMP in mitochondria following intermittent hypoxia may be a mechanism underlying this protection.

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein, and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547-554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

Polymorph Crystal Selection by n16, an Intrinsically Disordered Nacre Framework Protein

Framework proteins are a subclass of mollusk shell nacre-associated polypeptides that form supramolecular assemblies with β-chitin and other matrix proteins. These macromolecular assemblies manage the energetics of aragonite polymorph nucleation, and thus, there is keen interest in understanding the molecular characteristics of framework proteins. Here, we report the mineralization activity, oligomerization, and structural features of a recombinant framework nacre protein n16, isoform 3 (r-n16.3, Japanese pearl oyster Pinctada fucata). We find that r-n16.3 assembles in mineralization solutions to form spheroidal-fibril and mineralized thin film assemblies, in addition to spherical vaterite mineral deposits and aragonite single crystal deposits that possess unusual texture and layered morphologies. The oligomerization of r-n16.3 is spontaneous over the pH range 5–8.5, and protein particle sizes are observed to increase in radii when Ca(II) is present. Bioinformatics studies reveal that the r-n16.3 molecule is intrinsically disordered (random coil) and possesses residual α helix and β sheet structure. Experimentally, we confirmed that the secondary structure of apo-r-n16.3 assemblies is largely disordered (50% random coil, 20% β strand, 8% α helix). However, in the presence of high Ca(II) concentrations, we observe IDP disorder-to-order transformations that increase β turn structure and decrease random coil, α helix, and β strand contents. We conclude that r-n16.3 is an intrinsically disordered oligomeric nacre framework protein that nucleates vaterite and single crystal aragonite in vitro and possesses target-specific IDP disorder-to-order transformation capabilities in response to Ca(II).

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2(D307H) protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca(2+) buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca(2+). These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca(2+) activation.

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.

Humic substances and the water calcium content change the toxicity of malachite green

Summary Laboratory experiments were conducted to test interactive effects of calcium(Ca 2+ ) content and the presence of humic substance (HS) on malachite green (MAG)-induced toxicity in fish embryos and larvae by means of a semistatic 144-hembryo-larval-test with zebrafish (Danio rerio). Two kinds of reconstituted water samples were used to produce the test media by mixing salts into deionized water resulting in either hard water (›Ca ) HS), or soft water (flCa ) HS). By adding HS two additional test media were produced (›Ca + HS, flCa + HS). MAG was tested in concentrations of 0.05, 0.10, 0.15, 0.20, 0.25 mg L )1 . The toxicity ranking of MAG (mg L )1 ) to embryos based on 96-h-LC50 in the different test water samples is: ›Ca ) HS (0.061) > ›Ca + HS (0.123) ¼ flCa ) HS (0.12) ‡fl Ca + HS (0.134) and on 144-h-LC50 to larvae is: ›Ca ) HS (0.038) > ›Ca + HS (0.06) > flCa ) HS (0.077) ¼ flCa + HS (0.077). Mortality of all the groups was significantly different (P < 0.05). Increased Ca 2+ concentrations did not protect zebrafish embryos and larvae from MAG-induced toxicity. At high Ca 2+ conditions, the mortality of the embryos as well as of the larvae is reduced in the ›Ca + HS group relative to the ›Ca ) HS group. Thus, at high Ca 2+ conditions the HS does affect the MAG-induced mortality. The mechanism which causes the higher toxicity of MAG in the presence of higher Ca 2+ concentrations is poorly understood. A probable explanation could be the stimulation of the calcium-binding protein calmodulin as well as the calmodulin kinase II in cell membranes in the presence of high Ca 2+ concentrations.

C-erbB 2 Serum level as prognostic factor in hormonally treated advanced breast cancer patients

To investigate whether c-erbB 2 serum levels may be predictive of clinical response, progression-free and overall survival in postmenopausal women with advanced breast cancer hormonally treated, 265 patients enrolled in previous clinical trials were evaluated. C-erbB 2 serum levels were assessed before the start of treatment and in a subgroup of patients also at the first response evaluation. In addition, serum CA 15.3 levels were determined. The role of c-erbB 2 was investigated by means of multiple regression models in which both c-erbB 2 and CA 15.3 values were modelled as continuous variables together with other known prognostic factors. The failure probability tended to be higher in the presence of high c-erbB 2 levels, but the trend was not statistically significant; in contrast, significant results were obtained for progression-free survival (PFS, P <0.001) and overall survival (OS, P=0.014). The within-patient c-erbB 2 variation significantly predicted PFS ( P=0.006) and OS ( P=0.040). It is worth noting that c-erbB 2 and CA 15.3 baseline levels were significantly correlated and that the prognostic effect of c-erbB 2 tended to disappear in the presence of high CA 15.3 levels for PFS and OS.

The Slippage of the Ca2+ Pump and Its Control by Anions and Curcumin in Skeletal and Cardiac Sarcoplasmic Reticulum

Ca(2+) transport by sarcoplasmic reticulum (SR) ATPase occurs with an optimal coupling ratio of 2 Ca(2+) per ATP in pre-steady state. However, slippage of the pump and lower coupling ratios are observed in steady state. Slippage depends on the presence of high Ca(2+) in the lumen of SR vesicles and high nucleotide in the medium. Thereby, Ca(2+) and/or nucleotide-bound phosphoenzyme intermediates accumulate and undergo uncoupled cleavage, before vectorial translocation of bound Ca(2+) in the forward direction of the cycle or before productive reversal to ATP synthesis. Transport efficiency and coupling ratios are improved by reduction of nucleotide concentration in the presence of ATP regenerating systems and/or complexation of luminal Ca(2+) with phosphate or oxalate. Curcumin (1-5 microm) lowers the concentration of phosphate or oxalate required to reduce slippage of the Ca(2+) pump. Thereby, under appropriate conditions, curcumin favors kinetic flow, completion of productive cycles, and improvement of coupling ratios. The findings obtained with isolated SR vesicles suggest that slippage is an important phenomenon under prevailing conditions of muscle fibers in situ. Ca(2+) transport and its slippage can be improved by curcumin in cardiac as well as in skeletal SR, raising the possibility of pharmacological interventions to correct defective Ca(2+) homeostasis. Higher curcumin concentrations (5-30 microm), however, inhibit overall ATPase activity and Ca(2+) transport by interfering with phosphoenzyme formation with ATP or P(i).

Unravelling the Interaction of Thapsigargin with the Conformational States of Ca2+-ATPase from Skeletal Sarcoplasmic Reticulum

Preincubation of thapsigargin with sarcoplasmic reticulum vesicles in the presence of high Ca(2+) or the addition of high Ca(2+) to microsomal vesicles preincubated with thapsigargin in the absence of Ca(2+) allowed full enzyme phosphorylation by ATP. However, the enzyme activity was not protected by high Ca(2+) even when the samples were subjected to gel filtration before ATP addition. Our data indicate that: (i) the enzyme in the Ca(2+)-bound conformation can be stabilized in the presence of thapsigargin; (ii) the conformational transition from the Ca(2+)-free to the Ca(2+)-bound state can be elicited by Ca(2+) when thapsigargin is present; (iii) thapsigargin binding occurs whether or not the enzyme is in the presence of Ca(2+), and so a ternary complex enzyme-Ca(2+)-thapsigargin may be formed; (iv) thapsigargin can be dissociated from the enzyme with a slow kinetics after dilution under drastic conditions; (v) the kinetics of Ca(2+) binding is clearly slowed down by thapsigargin; and (vi) thapsigargin does not affect the hydrolysis rate of phosphorylating substrates when measured in the absence of Ca(2+), indicating that thapsigargin specifically inhibits the Ca(2+)-dependent activity.

The toxicity of the antiparasitic mixture FMC is changed by humic substances and calcium

An inexpensive mixture of the therapeutic agents malachite green (MAG), methylene blue (MB) and formalin (known as FMC) is used to control external fungal infections in fish eggs (e.g. Saprolegnia ssp., Achlya ssp.) in aquaculture and ornamental fish breeding. The treatment success as well as the toxicity of the chemicals is determined by the toxicity of each of the substances and synergistic effects among them. Moreover, toxicity and therapeutical index might be affected by water parameters such as the ion content and the presence of humic substances (HS). Four different test waters with combinations of low (0.2 mmol L−1) and high (2 mmol L−1) calcium (Ca), +HS (5 mg L−1 C) or –HS (0 mg L−1 C) were used to determine the toxicity of FMC to early life stages of zebrafish, Danio rerio (Hamilton-Buchanan, 1822, 1823). Thus, the test solutions differed only in the content of dissolved organic carbon, and/or the amount of Ca chloride, and, to a lesser extent, pH. Zebrafish eggs reaching the four- to eight-cell stage were exposed to different concentrations of FMC (10, 15, 22, 33, 50 µL L−1 FMC) for 144 h. Survival was highest in low Ca/+HS-solution. High Ca and HS did not better protect the eggs than HS alone, so that this group had the lowest survival. Survival of the ‘–HS-groups’ was intermediate. The toxicity of the mixture as well as the interaction with Ca and HS was mainly determined by MAG. MAG, the most toxic part of the mixture, is positively charged and competes with Ca for the negatively charged binding sites of the HS. In presence of high Ca, the binding sites of the HS are filled with Ca-ions. This causes lower binding of MAG to the HS. Thus, MAG binds to the juvenile fish and enhances the toxicity of the mixture. Therefore, the toxicity as well as the therapeutical index may be altered in the presence of HS and the Ca content of the water.

Ca 2+ overloading causes the negative inotropic effect of doxorubicin in myocytes isolated from guinea-pig hearts

We reported previously that conditions shown to increase Ca 2+ loading augment the negative inotropic effect of doxorubicin. To examine if the negative inotropic effect is caused by Ca 2+ overloading, doxorubicin-induced changes in diastolic and systolic Ca 2+ concentrations and twitch contractions were studied under altered Ca 2+ loading. Intracellular Ca 2+ concentrations ([Ca 2+] i) were monitored in fura-2-loaded myocytes isolated from the ventricular muscle of guinea-pig hearts. Twitch contractions were estimated from the shortening of myocytes. In myocytes incubated at 34°C in a medium containing 1.2 mM CaCl 2 (standard conditions), doxorubicin (100 μM) caused a significant decrease in diastolic length, and an increase in the amplitude of contraction. The positive inotropic effect of doxorubicin was followed by a negative effect. Concomitant with these changes in myocyte contractions, a monophasic increase in diastolic Ca 2+, and an increase and a subsequent decrease in the amplitude of Ca 2+ transients (peak [Ca 2+] i minus diastolic [Ca 2+] i) were observed. When the Ca 2+ load of myocytes was increased by an incubation at a low temperature (25°C) or in the presence of high Ca 2+ in the incubation medium (2.4 mM CaCl 2), diastolic [Ca 2+] i was elevated. Doxorubicin further increased diastolic [Ca 2+] i. Under these conditions, the doxorubicin-induced increase in the twitch contraction lasted a shorter period of time and the subsequent decrease in contraction was significantly enhanced. The peak [Ca 2+] i initially increased slightly and then decreased. Thus, the decrease in the amplitude of Ca 2+ transients, as well as myocyte contraction, was greater compared to the corresponding values observed under the standard conditions. These changes in the contraction and Ca 2+ transient developed with the same time course. The effects of the low-temperature incubation were antagonized by verapamil. These results indicate that the negative inotropic effect of doxorubicin results from a decrease in the amplitude of Ca 2+ transients caused by an increased diastolic [Ca 2+] i and a decreased peak [Ca 2+] i. These changes are likely to be caused by myocardial Ca 2+ overload. © 1997 Elsevier Science B.V. All rights reserved.

Factors Influencing the Effectiveness and Stability of Aqueous Lead Immobilization by Hydroxyapatite

The effects of Ca, K, Na, calcite, and EDTA on Pb immobilization by hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ; HA] were investigated. The stability of hydroxypyromorphite [Pb 10 (PO 4 ) 6 (OH) 2 ; HP] (a reaction product of HA with Pb) and the effectiveness of using HA to immobilize Pb in Pb-contaminated soils also were examined. Neither Na nor K affected Pb immobilization. Aqueous Ca and calcite slightly Inhibited Pb immobilization, but HA still reduced aqueous Pb > 97% in the presence of these species ; however, EDTA significantly reduced the effectiveness of HA to immobilize Pb from Pb-EDTA solutions, with only 24 to 31% resultant reduction in aqueous Pb. Hydroxypyromorphite was stable in the presence of HA up to 8 wk. Both higher Ca and lower P concentrations (following P removal by anion exchange resin) dissolved HP from a mixture of HA and HP, but the final aqueous Pb concentrations remained low ( 85%) of the Pb from the same mixture. Hydroxyapatite effectively immobilized >71% of the aqueous Pb in four PbHAsO 4 contaminated soils. The effectiveness of HA to immobilize aqueous Pb in the presence of Ca, K, Na, and calcite, and to immobilize aqueous Pb from Pb-contaminated soils, together with the stability of HP in the presence of high Ca and low P concentrations support our previous conclusion that HA has the potential to treat Pb contaminated water, soils, and wastes and that such a treatment can result in long-term in situ Pb stabilization ; however, HA did not effectively immobilize Pb, nor was HP, stable in the presence of high EDTA concentrations. Thus, HA treatment will be ineffective in EDTA rich environments.

CHLORTHALIDONE-MODULATED VASCULAR RESPONSES TO VASODILATOR STIMULI IN DOCA-SALT HYPERTENSIVE RATS

This study was carried out to test the effect of chlorthalidone (Chlor) treatment on the vascular responsiveness to acetylcholine (Ach) and adenosine disphosphate (ADP) under different extracellular calcium concentrations (Ca e 2+) in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Half of the DOCA-salt hypertensive rats were simultaneously given Chlor (8mgday −1, p.o.), after the twentieth day of DOCA, until the fortieth day. Mean arterial pressure (MAP) was reduced ( P<0.01) in DOCA-salt-Chlor (121±3mmHg) compared to DOCA-salt (175±3mmHg) rats. Chlor reduced the high plasma sodium of DOCA-salt to levels of the normotensive groups. The basal perfusion pressure of the isolated mesenteric vascular bed (MVB) was not modified by Ca e 2+changing or previous addition of ouabain. At normal and low Ca e 2+the vasodilator effect of Ach in the MVB was reduced ( P<0.01) in DOCA-salt and less ( P<0.05) in the salt-control rats. Chlor shifted the curve of DOCA-salt backward to control group curves. In high Ca e 2+the vasodilator responses to Ach were reduced ( P<0.01) both in normal-control and DOCA-salt+Chlor but not in salt-control and DOCA-salt group. The vasodilator effect of ADP to different Ca e 2+was similar in presence of normal and low Ca e 2+in normal-control rats and it was reduced ( P<0.01) in presence of high Ca e 2+in the DOCA-salt group. Chlor restored the responses to ADP to normal-control level. Under ouabain-treatment, the vasodilatation duration was reduced ( P<0.05) in all groups. It was shorter ( P<0.01) in DOCA-salt than in the other ones. Chlor completely abolished this difference. The MAP reduction in DOCA-salt rats by chlor could also be associated with a restored normal responsiveness of endothelial cells to endogenous vasodilator, for example Ach and ADP, since sodium nitroprusside did not exhibit differences among the various treatments. This diuretic agent seems to elicit its effect through a reduction in plasma sodium content of DOCA-salt hypertensive rats.).

The inositol 1,4,5-trisphosphate receptor: kinetic properties and regulation

Inositol 1,4,5-trisphosphate (Ins P 3) is a second messenger responsible for the mobilization of intracellular Ca 2+ after receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. Ins P 3 binds to a specific receptor located on the membrane of an intracellular compartment and opens a Ca 2+ channel causing the cytosolic Ca 2+ concentration to increase. Measurement of radiolabelled Ins P 3 binding and Ins P 3-induced Ca 2+ release in parallel experiments indicated that the liver Ins P 3 receptor exists in two main states: an active state (A) and an inactive one (I). The “I” form of the receptor is found in the presence of high Ca 2+ concentrations (above 1 μM). The binding properties of the “A” and the “I” states of the receptor have been characterized by analysing a membrane fraction enriched in Ins P 3 receptors. The inactive “I” state displays a high affinity ( K d = 2 nM) and slow rates of association and dissociation. The active state “A” of the receptor displays complex kinetic properties. The rate of association and the rate of dissociation of labelled Ins P 3 are rapid phenomena probably involving several components. The apparent K d for the Ins P 3 binding is about 40 nM in a low Ca 2+ medium. The affinity of the “A” state of the receptor is increased by Ca 2+ (at concentrations lower than 0.5 μM) and by thiol reagents. The increase of the affinity of the receptor is due to a decrease of the dissociation rate constants. This lowers the threshold such that Ca 2+ is released at lower concentrations of Ins P 3. These data indicate that the binding of Ins P 3 to its receptor is a complex phenomenon involving the transition among several states. These changes in the receptor properties may be involved in the mechanisms underlying the positive and negative feedback for the Ins P 3-induced Ca 2+ release found in several cell types.

Can calmodulin function without binding calcium?

Calmodulin is a small Ca 2+-binding protein proposed to act as the intracellular Ca 2+ receptor that translates Ca 2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca 2+-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca 2+; one does not bind detectable levels of 45Ca 2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca 2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca 2+.

An in vitro model of nephrocalcinosis using rabbit inner medullary collecting tubular cell culture

In order to investigate the pathogenesis of medullary nephrocalcinosis, rabbit inner medullary collecting duct cells were grown in media containing different Ca++, PTH and pH levels. It was found that high Ca++ (7.8mM) only reduced growth slightly and that crystalline deposits were found under the cells. This suggests that high Ca++ is not severely toxic to the cells but can lead to deposition of calcium beneath the basement membrane. PTH did not effect cell growth even in the presence of high Ca++ implying that it has an indirect effect on tubular cells in medullary nephrocalcinosis associated with hyperparathyroidism. In renal tubular acidosis these cells are subjected to a persistently high urinary pH and low interstitial pH. Raising the pH reduced the cell growth in normal Ca++ medium whereas lowering the pH increased cell growth in vitro. Our results show that nephrocalcinosis is not due to the direct effect of raised pericellular Ca++ or PTH alone and that persistently alkaline tubular fluid may play a role.