Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons

Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 microM, for 15 s)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 +/- 30 s, whereas recovery time was 216 +/- 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 (25 microM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.     

Increase of empty pool-activated Ca2+ influx using an intracellular Ca2+ chelating agent

We demonstrate here that stimulated 45Ca2+ influx in A7r5 vascular smooth muscle cells induced either by receptor activation with [Arg]8 vasopressin or by the SR-Ca(2+)-ATPase inhibitor thapsigargin was increased more than threefold if cells were preloaded with the intracellular calcium chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The increased influx is probably due to an attenuation of negative feedback of Ca2+ on its own entry accompanied by increased Ca2+ storage capacity of BAPTA-loaded cells leading to diminished cellular Ca2+ release. We propose that BAPTA preloading could be a useful approach to investigate receptor-induced Ca2+ influx.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.     

Changes in mitochondrial Ca2+ homeostasis in primary sensory neurons of diabetic mice

Changes in neuronal Ca2+ homeostasis were studied on freshly isolated dorsal root ganglion neurons of adult control mice and mice with streptozotocin (STZ)-induced diabetes. The cytoplasmic free Ca2+ concentration ([Ca2+]in) was measured using indo-1 based microfluorimetry. The participation of mitochondria in [Ca2+]in homeostasis was determined by investigation of changes which occurred after addition of mitochondrial protonophore (CCCP) to the extracellular solution. In control cells 10 microM CCCP applied before membrane depolarization induced an increase of the amplitude of depolarization-induced [Ca2+]in transients and disappearance of their delayed recovery, indicating the participation of mitochondria in fast uptake of Ca2+ ions from the cytosol during the peak of the transient and subsequent slow release them back during its decay. In diabetic animals the increase of the peak transient amplitude under the action of CCCP became diminished in small (nociceptive) neurons and the delayed elevation of [Ca2+]in disappeared in both large and small neurons. It is concluded that in diabetic conditions substantial changes occur in the Ca2+ homeostatic functions of mitochondria, manifested by decreased Ca2+ uptake in small neurons and depressed Ca2+ release into the cytosol in all types of neurons.     

Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.     

Simultaneous measurements of cytosolic and mitochondrial Ca2+ transients in HT29 cells

Loading of HT29 cells with the Ca2+ dye fura-2/AM resulted in an nonhomogeneous intracellular distribution of the dye. Cellular compartments with high fura-2 concentrations were identified by correlation with mitochondrial markers, cellular autofluorescence induced by UV, and dynamic measurement of autofluorescence after inhibition of oxidative phosphorylation. Stimulation with carbachol (10(-4) mol/liter) increased cytosolic, nuclear, and mitochondrial Ca2+ activity ([Ca2+]c, [Ca2+]n, and [Ca2+]m, respectively) measured by UV confocal and conventional imaging. Similar results were obtained with a prototype two-photon microscope (Zeiss, Jena, Germany) allowing for fura-2 excitation. The increase of [Ca2+]m lagged behind that of [Ca2+]c and [Ca2+]n by 10-20 s, and after removing the agonist, [Ca2+]m also decreased with a delay. A strong increase of [Ca2+]m occurred only when a certain threshold of [Ca2+]c (around 1 micromol/liter) was exceeded. In a very similar way, ATP, neurotensin, and thapsigargin increased [Ca2+]c and [Ca2+]m. Carbonyl cyanide p-trifluoromethoxyphenylhyrdrazone reversibly reduced the increase of [Ca2+]m. The source of the mitochondrial Ca2+ increase had intra- and extracellular components, as revealed by experiments in low extracellular Ca2+. We conclude that agonist-induced Ca2+ signals are transduced into mitochondria. 1) Mitochondria could serve as a Ca2+ sink, 2) mitochondria could allow the modulation of [Ca2+]c and [Ca2+]n signals, and 3) [Ca2+]m may serve as a stimulatory metabolic signal when a cell is highly stimulated.     

Fluorescence probe study of the lumenal Ca2+ of the sarcoplasmic reticulum vesicles during Ca2+ uptake and Ca2+ release

A limited amount of information is available about the lumenal Ca2+ kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+ quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+ uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+ ([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lum during uptake and release show the characteristics intrinsic to the SR Ca2+ pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+ release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lum measurement is that during the uptake reaction the free [Ca2+]lum showed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+ pump and lumenal proteins.     

Ca2+ in the dense tubules: a model of platelet Ca2+ load

In this work, we explored the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (Vmax) of the SERCA significantly correlated with FECa2+ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the Vmax of the SERCA (r=.462, P=.010) and the FECa2+ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca2+ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca2+ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca2+ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa2+ in the DT, and (2) a higher BP is associated with an increase in platelet Ca2+ load in human beings, expressed by a rise in the FECa2+ in the DT and the upregulation of SERCA activity.     

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.     

Saltatory propagation of Ca2+ waves by Ca2+ sparks

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.     

Effect of Ca2+ on the activity of mitochondrial NADP-specific isocitrate dehydrogenase from rabbit adrenals

Laparoscopic visualization techniques have improved dramatically over the last 5 years and have led to reassessment of the laparoscope for use in the staging of intraabdominal malignancy. One hundred sixty-two consecutive cases undergoing preoperative staging laparoscopy from January 1988 to December 1993 were reviewed in order to determine the value of laparoscopy as a staging tool. Indications for staging laparoscopy were predominantly hepatopancreaticobiliary (85%); however, other primaries such as stomach and colon were included. In 36% of cases information found at laparoscopy precluded resection and prevented unnecessary laparotomy. Additional information that was felt to be helpful in planning resection was found in 30% of cases. In 12% of cases unresectability was found only at the time of laparotomy and was missed by staging laparoscopy. We conclude that laparoscopy is a useful preoperative staging tool and can help avoid unnecessary laparotomy for intraabdominal malignancy in one-third of patients.     

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.