Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Interactions of physiological ligands with the Ca pump and Na/Ca exchange in squid axons

We have studied the interaction of physiological ligands other than Nai and Cai with the Ca pump and Na/Ca exchange in internally dialyzed squid axons. The results show the following. (a) Internal Mg2+ is an inhibitor of the Nao-dependent Ca efflux. At physiological Mg2+i (4 mM), the inhibition amounts to approximately 50%. The inhibition is partial and noncompetitive with Cai, and is not affected by Nai or ATP. The ATP-dependent uncoupled efflux is unaffected by Mgi up to 20 mM. Both components of the Ca efflux require Mg2+i for their activation by ATP. (b) At constant membrane potential, Ki is an important cofactor for the uncoupled Ca efflux. (c) Orthophosphate (Pi) activates the Nao-dependent Ca efflux without affecting the uncoupled component. Activation by Pi occurs only in the presence of Mg-ATP or hydrolyzable ATP analogues. Pi under physiological conditions has no effect on the uncoupled component; nevertheless, at alkaline pH, it inhibits the Ca pump, probably by product inhibition. (d) ADP is a potent inhibitor of the uncoupled Ca efflux. The Nao-dependent component is inhibited by ADP only at much higher ADP concentrations. These results indicate that (a) depending on the concentration of Ca2+i, Na+i Mg2+i, and Pi, the Na/Ca carrier can operate under a low- or high-rate regime; (b) the interactions of Mg2+i, Pi, Na+i, and ATP with the carrier are not interdependent; (c) the effect of Pi on the carrier-mediated Ca efflux resembles the stimulation of the Nao-dependent Ca efflux by internal vanadate; (d) the ligand effects on the uncoupled Ca efflux are of the type seen in the Ca pump in red cells and the sarcoplasmic reticulum.     

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

Studies on the energy-linked Ca2+ accumulation in pig heart mitochondria - role of Mg2'ons

Comparative intracellular distribution of Ca2+, Mg2+ and adenine nucleotides has been studied in pig heart by differential centrifugation or fractional extraction and has shown that Mg2+ and ATP are associated mainly with soluble fractions whereas Ca2+ and ADP are more tightly bound to subcellular structures. Ca2+ accumulation and Ca2+ stimulated respiration were studied in pig heart mitochondria under different energetic conditions in the absence or presence of phosphate. Ca2+ concentrations of about 1200 nmoles/mg protein inhibit Ca2+ accumulation, site I substrate oxidation and induce an efflux of mitochondrial Mg2+. These deleterious effects of Ca2+ on respiration occur even in the absence of phosphate or oxidizable substrate; they are completely prevented by ruthenium red only, and partially prevented by the addition of M2+ to the medium. The kinetics of Ca2+ uptake become of the sigmoidal type when Mg2+ is present. This cation strongly inhibits the rate of Ca2+ uptake in the presence of added phosphate and decreases the affinity of Ca2+ for its transport system. In the absence of phosphate, Mg2+ has no effect on Ca2+ uptake. The possible physiological implications of these findings are discussed     

The Ca2+-releasing messenger NAADP, a new player in the nervous system.

Many physiological processes are controlled by a great diversity of Ca2+ signals. Within cell, Ca2+ signals depend upon Ca2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca2+-releasing messengers which may interact and shape complex Ca2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons.     

Redox regulation of RyR-mediated Ca2+ release in muscle and neurons

Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca(2+)-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well.     

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.     

Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that is for the largest part expressed in the endoplasmic reticulum. Its precise subcellular localization is an important factor for the correct initiation and propagation of Ca2+ signals. The relative position of the IP3Rs, and thus of the IP3-sensitive Ca2+ stores, to mitochondria, nucleus or plasma membrane determines in many cases the physiological consequences of IP3-induced Ca2+ release. Most cell types express more than one IP3R isoform and their subcellular distribution is cell-type dependent. Moreover, it was recently demonstrated that depending on the physiological status of the cell redistribution of IP3Rs and/or of IP3-sensitive Ca2+ stores could occur. This indicates that the cell must be able to regulate not only IP3R expression but also its distribution. The various proteins potentially determining IP3R localization and redistribution will therefore be discussed.     

Blockade of cardiac sarcoplasmic reticulum K+ channel by Ca2+: two-binding-site model of blockade.

Potassium countercurrent through the SR K+ channel plays an important role in Ca2+ release from the SR. To see if Ca2+ regulates the channel, we incorporated canine cardiac SR K+ channel into lipid bilayers. Calcium ions present in either the SR lumenal (trans) or cytoplasmic (cis) side blocked the cardiac SR K+ channel in a voltage-dependent manner. When Ca2+ was present on both sides, however, the block appeared to be voltage independent. A two-binding site model of blockade by an impermeant divalent cation (Ca2+) can explain this apparent contradiction. Estimates of SR Ca2+ concentration suggest that under physiological conditions the cardiac SR K+ channel is partially blocked by Ca2+ ions present in the lumen of the SR. The reduction in lumenal [Ca2+] during Ca2+ release could increase K+ conductance.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

Calcimimetic and calcilytic drugs: just for parathyroid cells?

The cell surface calcium receptor (Ca2+ receptor) is a particularly difficult receptor to study because its primary physiological ligand, Ca2+, affects numerous biological processes both within and outside of cells. Because of this, distinguishing effects of extracellular Ca2+ mediated by the Ca2+ receptor from those mediated by other mechanisms is challenging. Certain pharmacological approaches, however, when combined with appropriate experimental designs, can be used to more confidently identify cellular responses regulated by the Ca2+ receptor and select those that might be targeted therapeutically. The Ca2+ receptor on parathyroid cells, because it is the primary mechanism regulating secretion of parathyroid hormone (PTH), is one such target. Calcimimetic compounds, which active this Ca2+ receptor and lower circulating levels of PTH, have been developed for treating hyperparathyroidism. The converse pharmaceutical approach, involving calcilytic compounds that block parathyroid cell Ca2+ receptors and stimulate PTH secretion thereby providing an anabolic therapy for osteoporosis, still awaits clinical validation. Although Ca2+ receptors are expressed throughout the body and in many tissues that are not intimately involved in systemic Ca2+ homeostasis, their physiological and/or pathological significance remains speculative and their value as therapeutic targets is unknown.     

Glucose-induced buffering of cytoplasmic Ca2+ in the pancreatic beta-cell--an artifact or a physiological phenomenon?

The effects of stimulated metabolism on the cytoplasmic Ca2+ concentration (Ca2+i) of insulin-releasing pancreatic beta-cells were studied. When the glucose concentration was increased from 5 to 20 mM, some cell preparations responded with initial lowering of Ca2+i followed by a rise, whereas Ca2+i only increased in others. After prolonged exposure to 5 or 10 mM of the sugar, depolarization with high concentrations of sulfonylurea or K+ caused rapid increases of Ca2+i. However, when subsequently raising glucose to 20 mM there were pronounced temporary decreases of Ca2+i. Marked Ca2+i reducing effects were also obtained after prolonged exposure to 20 mM glucose, when metabolism was augmented further by exposure to leucine or beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. The results indicate that buffering of Ca2+i is not an artifact but may have physiological significance.     

Functional correlation between cell adhesive properties and some cell surface proteins

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+- dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+- dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.     

Mechanical compression influences intracellular Ca2+ signaling in chondrocytes seeded in agarose constructs.

Ca2+ signaling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. In this study, a well-characterized model system utilizing bovine articular chondrocytes embedded in 4% agarose constructs was used to investigate the effect of physiological mechanical compressive strain applied after 1 and 3 days in culture. The intracellular Ca2+ concentration was measured by use of the ratiometric Ca2+ indicator indo 1-AM and confocal microscopy. A positive Ca2+ response was defined as a percent increase in Ca2+ ratio above a preset threshold. A significantly greater percentage of cells exhibited a positive Ca2+ response in strained constructs compared with unstrained controls at both time points. In strained constructs, treatment with either Ga3+ or EGTA significantly reduced the number of positive Ca2+ responders compared with untreated controls. These results represent an important step in understanding the physiological role of intracellular Ca2+ in chondrocytes under mechanical compression.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) and Ca2+ mobilization

Many physiological processes are controlled by a great diversity of Ca2+ signals that depend on Ca2+ entry into the cell and/or Ca2+ release from internal Ca2+ stores. Ca2+ mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca2+ release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP3 and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca2+ stores. Activation of the NAADP-sensitive Ca2+ channels evokes complex changes in cytoplasmic Ca2+ levels by means of channel chatter with other intracellular Ca2+ channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca2+ signaling.     

Mitochondria as regulators of stimulus-evoked calcium signals in neurons

An important challenge in the study of Ca2+ signalling is to understand the dynamics of intracellular Ca2+ levels during and after physiological stimulation. While extensive information is available regarding the structural and biophysical properties of Ca2+ channels, pumps and exchangers that control cellular Ca2+ movements, little is known about the quantitative properties of the transporters that are expressed together in intact cells or about the way they operate as a system to orchestrate stimulus-induced Ca2+ signals. This lack of information is particularly striking given that many qualitative properties of Ca2+ signals (e.g. whether the Ca2+ concentration within a particular organelle rises or falls during stimulation) depend critically on quantitative properties of the underlying Ca2+ transporters (e.g. the rates of Ca2+ uptake and release by the organelle). This monograph describes the in situ characterization of Ca2+ transport pathways in sympathetic neurons, showing how mitochondrial Ca2+ uptake and release systems define the direction and rate of net Ca2+ transport by this organelle, and how the interplay between mitochondrial Ca2+ transport and Ca+2 transport across the plasma membrane contribute to depolarization-evoked Ca2+ signals in intact cells.