A hypothesis for the local control of osteoclast function by Ca2+, nitric oxide and free radicals

Several important conclusions have recently emerged fromin vitro studies on the resorptive cell of bone, the osteoclast. First, it has been established that osteoclast function is modulated locally, by changes in the local concentration of Ca²⁺ caused by hydroxyapatite dissolution. It is thought that activation by Ca²⁺ of a surface membrane Ca²⁺ receptor mediates these effects, hence providing a feedback control. Second, a number of molecules produced locally by the endothelial cell, with which the osteoclast is in intimate contact, have been found to affect bone resorption profoundly. For instance, the autocoid nitric oxide strongly inhibits bone resorption. Finally, reactive oxygen species have been found to aid bone resorption and enhance osteoclastic activity directly. Here, we will attempt to integrate these control mechanisms into a unified hypothesis for the local control of bone resorption.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Effect of hypercholesterolemia on myocardial function in New Zealand white rabbits

Although hypercholesterolemia is a well-known risk factor for atherosclerosis, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of this study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed standard chow (control group) and another 15 were fed a cholesterolenriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rates. The whole-cell calcium current recording, and mRNA and protein levels of cellular calcium-handling proteins were also analyzed. With 2 mM Ca²⁺ and stimulation at 3 Hz, the contractile force of HC strips was less than that of the controls (3.63±0.20 vs. 4.61±0.50 mN, p<0.05). The time to peak tension was longer for HC strips (93.3±2.16 vs. 82.2±2.81 ms, p < 0.05). The peak L-type calcium inward current density was slightly higher in HC myocytes but did not reach statistical significance (–14.90±0.94 vs. –12.44±0.84 pA/pF, p=0.15). The mRNA level of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), normalized to GAPDH, was significantly lower in the HC than that in the control group (2.85±0.14 vs. 7.67±0.67, p<0.05), as was the ryanodine receptor (RyR; 0.42±0.06 vs. 0.71±0.13, p<0.05). The mRNA of the Na⁺/Ca²⁺ exchanger (NCX) was statistically higher in the HC group (0.90±0.12 vs. 0.48±0.05, p<0.05). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of the NCX increased. The protein expression of the dihydropyridine receptor was similar between these two groups. We concluded that hypercholesterolemia results in suppression of the maximal contractile function and in a longer systolic contractile time course. These changes may partially be mediated through a decrease in SERCA and RyR but an increase in NCX expression.     

Excitation-contraction coupling in a pre-vertebrate twitch muscle: The myotomes of Branchiostoma lanceolatum

Summary The segmented trunk muscle (myotome muscle) of the lancelet (Branchiostoma lanceolatum), a pre-vertebrate chordate, was studied in order to gain information regarding the evolution of excitation-contraction (EC) coupling.Myotome membrane vesicles could be separated on isopycnic sucrose gradients into two main fractions, probably comprising solitary microsomes and diads of plasma membrane and sarcoplasmic reticulum, respectively. Both fractions bound the dihydropyridine PN 200/110 and the phenylalkylamine (–)D888 (devapamil) while specific ryanodine binding was observed in the diad preparation only. Pharmacological effects on Ca²⁺ currents measured under voltage-clamp conditions in single myotome fibers included a weak block by the dihydropyridine nifedipine and a shift of the voltage dependences of inactivation and restoration to more negative potentials by (–)D888. After blocking the Ca²⁺ current by cadmium in voltage-clamped single fibers, the contractile response persisted and a rapid intramembrane charge movement could be demonstrated. Both responses exhibited a voltage sensitivity very similar to the one of the voltage-activated Ca²⁺ channels.Our biochemical and electrophysiological results indicate that the EC coupling mechanism of the protochordate myotome cell is similar to that of the vertebrate skeletal muscle fiber: Intracellular Ca²⁺ release, presumably taking place via the ryanodine receptor complex, is under control of the cell membrane potential. The sarcolemmal Ca²⁺ channels might serve as voltage sensors for this process.We thank Drs. H.Ch. Lüttgau and L.M.G. Heilmeyer, Jr. for stimulating discussions during the work, Dr. N.R. Brandt for helpful suggestions, and Drs. A.H. Caswell and M. Michalak for their generous gifts of antibodies. We also thank Ms. P. Goldmann, Mr. R. Schwalm, and Mr. U. Siemen for technical support and Ms. E. Linnepe for editorial help. This work was supported by grant G1 72/1-5 of the Deutsche Forschungsgemeinschaft. R. Benterbusch was recipient of a scholarship by the Studienstiftung des Deutschen Volkes.     

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.     

Calcium and inositol trisphosphate receptors

Work from the authors' laboratory is supported by the Wellcome Trust, and the Medical, and Agricultural and Food Research Councils. CWT is a Lister Institute Research Fellow.     

The effect of boromycin on the Ca2+ homeostasis

A boron-containing antibiotic, boromycin (BM), was found to influence the Ca²⁺ homeostasis in both excitable and non-excitable cells. In non-excitable cells (human erythrocytes and leucocytes) it inhibited the resting passive⁴⁵Ca²⁺ transport in 10^–6–10^–5 mol/L concentrations. In human erythrocytes, the passive ⁴⁵Ca²⁺ transport induced by the presence of 1 mmol/L NaVO₃ was inhibited by boromycin (90% inhibition) as well. The inhibitory effect of BM on the NaVO₃-induced passive ⁴⁵Ca²⁺ transport was diminished in the presence of inhibitory concentrations of nifedipine (10 mol/L – 60% inhibition) or of those of K⁺ _o (75 mmol/L – 20% inhibition). On the other hand, in rat brain synaptosomes, and rat cardiomyocytes, BM stimulated the passive ⁴⁵Ca²⁺ transport in resting cells at similar concentrations. In rat cardiomyocytes the stimulation was transient. The stimulatory effect on the passive ⁴⁵Ca²⁺ transport in rat brain synaptosomes was accompanied with the increase of cytoplasmic Ca²⁺ concentration measured by means of the entrapped fluorescent Ca²⁺ chelator fura-2. The stimulatory effect of BM was diminished when synaptosomes were pre-treated with veratridine (10 mol/L) which itself stimulated the passive ⁴⁵Ca²⁺ transport. At saturating concentrations of veratridine, no stimulatory effect of BM was observed. These results could be explained by the indirect interaction of BM with both Ca²⁺ and Na⁺ transport systems via transmembrane ionic gradients of monovalent cations and could be useful in determining whether the cells belong to excitable, or non-excitable cells.     

Single channel function of inositol 1,4,5-trisphosphate receptor type-1 and -2 isoform domain-swap chimeras

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893-3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Specific inhibitors of intracellular Ca2+ transport ATPases

Support from the National Institutes of Health and the American Heart Association is gratefully acknowledged.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

Vasodilative effect of perillaldehyde on isolated rat aorta

The vasodilative effect of perillaldehyde, one of the major oil components in Perilla frutescens BRITTON, was studied using isolated rat aorta. Perillaldehyde at final concentrations of 0.01 to 1 mM showed dose-dependent relaxation of the aorta contracted by treatment with prostaglandin F2alpha or norepinephrine. Neither the presence of NG-nitro-L-arginine methyl ester nor removal of the aortic endothelium affected the vasodilatation, suggesting that perillaldehyde exerts a direct effect on vascular smooth muscle cells. The vasodilative effect of perillaldehyde was not inhibited by pretreatment with a beta-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethylammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide). However, perillaldehyde showed contrasting effects on vasodilatation of the aorta contracted by an influx of extracellular Ca2+ - perillaldehyde caused little vasodilatation on the aorta contracted by the Ca2+ ionophore A23187, while it inhibited the vasoconstriction induced by treatment with high-concentration K+, which dominantly opened the voltage-dependent Ca2+ channel. These results suggest that the vasodilative effect of perillaldehyde is derived from blocking the Ca2+ channels.     

Physical coupling between ryanodine receptor-calcium release channels

Ryanodine receptor-calcium release channels play a pivotal role in the calcium signaling that mediates muscle excitation-contraction coupling. Their membrane organization into regular patterns, functional gating studies and theoretical analysis of receptor clustering have led to models that invoke allosteric interaction between individual channel oligomers as a critical mechanism for control of calcium release. Here we show that in reconstituted "checkerboard-like" lattices that mimic in situ membrane channel arrays, each oligomer is interlocked physically with four adjacent oligomers via a specific domain-domain interaction. Direct physical coupling between ryanodine receptors provides structural evidence for an inter-oligomer allosteric mechanism in channel regulation. Therefore, in addition to established cytosolic and luminal regulation of function, these observations indicate that channel-channel communication through physical coupling provides a novel mode of regulation of intracellular calcium release channels.