Regulation of [Ca2+]c oscillations by plasma membrane Ca2+ fluxes: a role for natriuretic peptides.

We have investigated the effects of natriuretic peptides on oscillations in [Ca(2+)](c) (cytosolic free Ca(2+) concentration) in two different cell types: freshly isolated rat hepatocytes and the ECV304 cell line. Our data, from both cell types, suggest that natriuretic peptides modulate the frequency of [Ca(2+)](c) oscillations through alterations in plasma membrane Ca(2+) fluxes. Here, we review evidence for a role for plasma membrane Ca(2+) fluxes in the control of the frequency of [Ca(2+)](c) oscillations. We describe a hypothetical mechanism through which this might be achieved. We propose a physiological role for regulated control of Ca(2+) efflux in modulating [Ca(2+)](c) oscillations.

A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle.

We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.

Synergy between Staphylococcus aureus and Pseudomonas aeruginosa in a rat model of complex orthopaedic wounds.

BACKGROUND: We observed an interaction in animals inoculated concomitantly with Staphylococcus aureus and Pseudomonas aeruginosa during a study of the efficacy of surfactants for disinfection of orthopaedic wounds. This led us to investigate whether synergy could be demonstrated between Staphylococcus aureus and Pseudomonas aeruginosa in a rat model of complex orthopaedic wounds. METHODS: A wire was implanted into the spinous process of a lumbar vertebra of Sprague-Dawley rats through a dorsal incision. Animals were divided into two groups: group one was inoculated with either Staphylococcus aureus or Pseudomonas aeruginosa, and group two received a polymicrobial inoculation with both test organisms in varying concentrations. After inoculation, the wounds were irrigated and closed. On postoperative day 14, all animals were killed and specimens from the wounds were cultured. The number of colony-forming units (CFU) of Staphylococcus aureus or Pseudomonas aeruginosa needed to cause infection in 50% of the animals (ID50) was determined with use of the Reed-Muench method. The infection rate associated with each inoculum combination was calculated, and the two groups were compared. RESULTS: The ID50 was 2.8 x 10(4) CFU for Staphylococcus aureus and 4.8 x 10(5) CFU for Pseudomonas aeruginosa. The combination of 10(3) CFU of Staphylococcus aureus with low concentrations (10(2), 10(3), or 10(4) CFU) of Pseudomonas aeruginosa yielded infection rates that were higher than those found with either organism alone at the same concentrations. The combination of 10(3) CFU of Staphylococcus aureus and 10(3) CFU of Pseudomonas aeruginosa yielded a 75% infection rate, which was significantly higher (p = 0.004) than that associated with 10(3) CFU of either organism alone. As the Pseudomonas aeruginosa concentration was increased (to 10(5), 10(6), and 10(7) CFU), this trend reversed, and the infection rate decreased to 33% (p = 0.004). Low concentrations of Pseudomonas aeruginosa (0 to 10(5) CFU) combined with 10(6) CFU of Staphylococcus aureus yielded infection rates ranging from 83% to 100%. At the higher concentrations of Pseudomonas aeruginosa (10(6) and 10(7) CFU), however, the infection rate again decreased, to 33% (p = 0.005). Only Staphylococcus aureus was isolated from the cultures of the specimens from the animals that had received a polymicrobial inoculum. CONCLUSIONS: Synergy between Staphylococcus aureus and Pseudomonas aeruginosa was demonstrated when low levels of each organism were present in the wound. As the Pseudomonas aeruginosa concentration was increased, the infection rates fell well below what would be anticipated, suggesting that low concentrations of Pseudomonas aeruginosa enhance the ability of Staphylococcus aureus to cause infection in this orthopaedic wound model. At the same time, the presence of Staphylococcus aureus in the ratios tested decreased the rate of infection by Pseudomonas aeruginosa. CLINICAL RELEVANCE: Staphylococcus aureus is a pathogen commonly seen in orthopaedic patients. The pathogenicity of Staphylococcus aureus was shown to be increased in the presence of anaerobic bacteria. This study is the first one that we are aware of that demonstrated synergy between Staphylococcus aureus and Pseudomonas aeruginosa, at low concentrations, in a wound model while at the same time showing that Staphylococcus aureus lowers the rate of Pseudomonas aeruginosa infection.

Activation of the particulate and not the soluble guanylate cyclase leads to the inhibition of Ca2+ extrusion through localized elevation of cGMP.

We examined whether localized increases in cytosolic cGMP have distinct regulatory effects on the concentration of cytosolic free Ca(2+) in ECV304 cells. Stimulation of the particulate guanylate cyclase by brain-type natriuretic peptide in fura-2-loaded cells caused a profound potentiation of the ATP-stimulated and thapsigargin-stimulated rise in cytosolic free Ca(2+). This effect is mediated by the inhibition of Ca(2+) extrusion via the plasma membrane Ca(2+)-ATPase pump. Furthermore, the addition of brain-type natriuretic peptide caused the partial inhibition of cation influx in ATP-stimulated cells. In contrast, elevation of cytosolic cGMP by activation of the soluble guanylate cyclase induced by the addition of sodium nitroprusside causes an increased reuptake of Ca(2+) into the intracellular stores without affecting cation influx or Ca(2+) efflux. Thus, localized pools of cGMP play distinct regulatory roles in the regulation of Ca(2+) homeostasis within individual cells. We define a new role for natriuretic peptides in the inhibition of Ca(2+) efflux that leads to the potentiation of agonist-evoked increases in cytosolic free Ca(2+).

Elevation of mitochondrial calcium by ryanodine-sensitive calcium-induced calcium release.

Calcium is an important regulator of mitochondrial function. Since there can be tight coupling between inositol 1,4, 5-trisphosphate-sensitive Ca(2+) release and elevation of mitochondrial calcium concentration, we have investigated whether a similar relationship exists between the release of Ca(2+) from the ryanodine receptor and the elevation of mitochondrial Ca(2+). Perfusion of permeabilized A10 cells with inositol 1,4, 5-trisphosphate resulted in a large transient elevation of mitochondrial Ca(2+) to about 8 microm. The response was inhibited by heparin but not ryanodine. Perfusion of the cells with Ca(2+) buffers in excess of 1 microm leads to large increases in mitochondrial Ca(2+) that are much greater than the perfused Ca(2+). These increases, which average around 10 microm, are enhanced by caffeine and inhibited by ryanodine and depletion of the intracellular stores with either orthovanadate or thapsigargin. We conclude that Ca(2+)-induced Ca(2+) release at the ryanodine receptor generates microdomains of elevated Ca(2+) that are sensed by adjacent mitochondria. In addition to ryanodine-sensitive stores acting as a source of Ca(2+), Ca(2+)-induced Ca(2+) release is required to generate efficient elevation of mitochondrial Ca(2+).

Diadenosine polyphosphates are largely ineffective as agonists at natively expressed P2Y(1) and P2Y(2) receptors on cultured human saphenous vein endothelial cells.

The diadenosine polyphosphates are a group of long-lasting compounds, released into the bloodstream by platelet degranulation. They mediate endothelium-dependent vasodilatation in several animal vascular systems via P2Y receptors coupled to increases in cytoplasmic calcium ([Ca(2+)](c)). However, there is little evidence of diadenosine-mediated vasodilatation in the human vasculature, and a direct interaction with natively expressed P2Y receptors on human endothelium has not been demonstrated. We have therefore studied the effects of diadenosines on primary cultures of human saphenous vein endothelial cells (HSVECs) and related this to the expression of P2Y receptors. HSVECs were loaded with the calcium-sensitive dye fura-2, and nucleotide-stimulated [Ca(2+)](c) responses were recorded. HSVECs responded to 10 microM UTP, ATP and 2-methylthio-ATP but not to UDP. Consistent with the recorded [Ca(2+)](c) responses, RT-PCR analysis of HSVEC RNA amplified specific products for the P2Y(1) and P2Y(2) receptors but not the P2Y(4) and P2Y(6) receptors. HSVECs responded to Ap(3)A with a rise in [Ca(2+)](c), but none of the other diadenosines tested elicited a response. Therefore natively expressed human P2Y(1) and P2Y(2) receptors are insensitive to diadenosine polyphosphates with the exception of Ap(3)A. We would therefore predict that the diadenosine polyphosphates have only a limited vasodilatory role in human saphenous veins.

Acute myeloblastic leukaemia: graft-versus-host and graft-versus-leukaemia responses to autologous IL-2 activated lymphocytes in rapid and slow disease.

Thirteen adults with acute myeloblastic leukaemia (AML) in early 2nd complete remission (CR) were treated with recombinant interleukin-2 (IL-2) and autologous IL-2-activated peripheral blood lymphocytes (LAK cells). All 13 developed IL-2-induced in vitro lymphocytoxicity against K562 and Daudi target cells. After seven years' follow-up, there was no overall improved survival compared with a historical control group treated with chemotherapy alone. However 7/13 patients developed T-cel-associated cutaneous graft-versus-host disease (GVHD), and 4/4 of these tested showed in vitro evidence of a T-cell-mediated graft-versus-leukaemia (GVL) effects. These had significantly longer 2nd CRs and survived longer. More lymphocytes were harvested and more LAK cells were reinfused in these seven cases. Since these patients also had longer 1st CRs, their GVL response to IL-2/LAK cells could be a feature of slowly progressive disease.

Fluorescent measurement of [Ca2+]c. Basic practical considerations.

Over the last decade the range of fluorescent indicators for Ca2+ has increased dramatically so that there are now a host of probes available. Each may offer particular advantages depending on the design of the experiment and the fluorometric equipment available. Careful choice of the indicator is therefore central to achieve a successful outcome. The probe that is chosen will, of course, depend on the aims of the experiment, on how the indicator will be introduced into the cell(s), and on the excitation source and detection equipment that are available. I hope that this chapter will not only help investigators choose the most appropriate indicator but, in addition, give an insight into what can be achieved using fluorescent Ca2+ indicators.

Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304.

1. To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ([Ca2+]c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists. 2. Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 microM for ATP, 2.5 microM for UTP and 14 microM for adenosine-5'-O-(3-thio)triphosphate (ATPgammaS). EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbetaS) and AMP were 0.5 microM, 3.5 microM, 15 microM and 4.7 microM respectively, but maximal [Ca2+]c responses were less than those produced by a maximal addition of ATP/UTP. ECV304 cells were unresponsive to UDP and beta,gamma,methyleneATP. 3. Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor. However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor. 4. ECV304 [Ca2+]c responses to 2-methylthioATP were inhibited in the presence of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas [Ca2+]c responses to UTP were unaffected by this treatment. 5. ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in [Ca2+]c. Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase. 6. ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A. In addition, ECV304 cells respond to AMP with increases in [Ca2+]c via an as yet uncharacterized receptor.

Okadaic acid induces the release of Ca2+ from intracellular stores in ECV304 endothelial cells.

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca2+ ([Ca2+]c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca2+]c both in the presence and absence of extracellular Ca2+. In the absence of extracellular Ca2+, depletion of the intracellular Ca2+ stores with either ATP (1 microM) or thapsigargin (100 nM) prevented any further release of Ca2+ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca2+), a prior release of Ca2+ induced by okadaic acid reduced the magnitude of the response to ATP (1 microM). Taken together these observations indicate that okadaic acid induces Ca2+ release from the agonist-sensitive pool. The okadaic acid-induced Ca2+ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca2+]c oscillations, which at their peak resulted in 40-100% cells, at any one time, having an elevated [Ca2+]c. The response appeared to propagate between adjacent cells and the elevation of [Ca2+]c appeared initially in the cell periphery. In adherent cells, the release of Ca2+ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca2+ from the agonist- and thapsigargin-sensitive intracellular stores.

Expression of slime interferes with in vitro detection of host protein receptors of Staphylococcus epidermidis.

We hypothesized that slime may mask bacterial molecules important in the attachment of Staphylococcus epidermidis to inanimate surfaces. In support of this hypothesis, we found that slime-negative strains attached significantly better to fibrinogen or fibronectin than the parent strains and exhibited greater surface hydrophobicity. Comparable results were obtained with 53 clinical isolates.

Modulation of mitochondrial Ca2+ in ECV304 endothelial cells by agents which elevate cAMP.

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca2+]m in response to agonist stimulation is dependent on Ca2+ influx, i.e. an ATP-induced sustained increase in [Ca2+]c results in a slow-onset, sustained elevation in [Ca2+]m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753-10759]. In this study, we have investigated the effect of raising cAMP on ATP-evoked elevations in both [Ca2+]m and [Ca2+]c by: (i) activating adenylate cyclase with the forskolin analogue--forskolin 6-[3'-(N,N-dimethylaminopropionyl)]-HCl (1 microM) (FA); (ii) addition of membrane permeable dibutyryl-cAMP (100 microM) (dbcAMP); and (iii) a combination of FA plus inhibition of cAMP phosphodiesterase using RO-20-1724 (17.5 microM) (RO);. We have found that protocols aimed at elevating cAMP significantly reduce the ATP-evoked (1-10 microM) rise in [Ca2+]m (n = 14); however, the [Ca2+]c response to ATP was not affected (n = 33). This new evidence shows that a second messenger system, other than Ca2+ itself, may influence [Ca2+]m changes in response to agonist stimulation.

Purification and characterization of the staphylococcal slime-associated antigen and its occurrence among Staphylococcus epidermis clinical isolates.

The Staphylococcus epidermidis slime-associated antigen (SAA) was purified and characterized. N-Acetyl-glucosamine accounted for 70% of the dry weight of SAA, which was immunolocalized on the ruthenium red-positive material produced by slime-positive strains. A total of 59% of slime-producing S. epidermidis clinical isolates expressed SAA, while the phenotype slime- SAA+ was never recovered.

A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells.

By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells < 4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with < 6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions.

Graft-versus-host disease following interleukin-2/lymphokine-activated killer (LAK) cell immunotherapy in a patient with acute myelogenous leukaemia in second complete remission: autologous LAK cells following allogeneic bone marrow transplantation are donor-derived.

A 48-year-old man was treated by allogeneic bone marrow transplantation (BMT) in first remission of M4 acute myelogenous leukaemia (AML). He experienced no graft-versus-host disease (GVHD) and 7 months later he relapsed. Following further chemotherapy, he entered a second complete remission; however, he refused a further allogeneic or autologous BMT but agreed to immunotherapy with interleukin-2 and autologous lymphokine-activated killer (LAK) cells. He tolerated this treatment well but went on to develop grade II skin GVHD. Polymerase chain reaction studies of DNA microsatellites of the autologous LAK cells showed that they were of donor origin. The patient remained well for 9 months until, immediately following the introduction of prednisolone for his persistent GVHD, he relapsed. He declined further active treatment and died 5 months later. The case shows that IL-2/LAK cells can be safely given to patients who have experienced no GVHD following allo-BMT and are likely to be effective through an ongoing graft-versus-leukaemia effect.

Platelet Membrane Glycoprotein IIb/IIIa has Sequence Homologies with Human Virus Proteins and Synthetic Viral Peptides Inhibit Anti-GPIIb/IIIa Antibodies in Autoimmune Thrombocytopenic Purpura.

Human platelet GP IIb/IIIa and common human viruses showed sequence homologies of up to 220 amino acids. High scoring homologies were found in Herpes Simplex, Varicella Zoster, Epstein-Barr virus, Adenovirus and Cytomegalovirus, all of which cause lifelong latent infections. Further high scoring sequences were found in Measles, Mumps and Rubella, which are sporadically associated with acute autoimmune thrombocytopenic purpura (AITP). Lower scoring homologies were found in Parvovirus, coxsackie B and Human Immunodeficiency Virus. There were frequent homologies to known autoantibody-binding epitopes in the cysteine-rich and intracytoplasmic regions of GP IIb/IIIa, but also with the RGD-binding and calcium-binding regions, and with the nascent GP signal peptide which is not expressed in the functional glycoprotein. Peptides representing the 48 highest scoring viral sequences were synthesised in vitro, and 7 of these viral peptides were shown to inhibit the serum autoantibodies of adults with chronic AITP. The pattern and degree of autoantibody inhibition varied from patient to patient, was concentration dependent and distinct for each peptide. This suggests that polyclonal GP IIb/IIIa autoantibodies are directed to different GP epitopes and are cross reactive in different patients to different viral proteins in different viruses. The results suggest that human viruses have a role in the aetiology of AITP via molecular mimicry of platelet GP IIb/IIIa, and that chronic auto immunity may be related to a persistent antigenic stimulus from lifelong latent viral infections.

Autoimmune thrombocytopenia: anti-glycoprotein IIb/IIIa auto antibodies are reduced after human anti-D immunoglobulin treatment.

In chronic autoimmune thrombocytopenic purpura (AITP), an indirect monoclonal antibody immobilised platelet antigen (MAIPA) assay detected serum glycoprotein (GP) IIb/IIIa antibodies in 16/39 (41%) cases. In patients with clinically active AITP, a direct MAIPA assay detected platelet-associated GP IIb/IIIa kantibodies in 8/13 (62%) cases. Platelet bound and serum antibody concentrations suggested a high antibody affinity for platelet membrane glycoprotein IIb/IIIa. Five AITP patients with platelet associated glycoprotein IIb/IIIa antibodies were treated with intravenous anti D immunoglobulin. All showed an increase in platelet counts and a decrease in platelet associated autoantibody. These responses could be due to immunosuppressive anti-idiotype antibodies in anti D immunoglobulin.

Hyperparathyroidism, platelet intracellular free calcium and hypertension in chronic renal failure.

To investigate possible relationships between hyperparathyroidism, alterations in intracellular free calcium concentration ([Ca2+]i) and hypertension in chronic renal failure, serum concentrations of intact parathyroid hormone (PTH) were measured by two-site immunometric assay, and platelet ([Ca2+]i) was assessed using the fluorescent indicator fura-2. Thirty-six patients with chronic renal failure were studied, 10 with normal serum PTH concentrations (mean 8.0 +/- 0.6 pmol/liter), 17 with elevated serum PTH (35.0 +/- 7.2 pmol/liter) and 9 patients with elevated PTH (36.2 +/- 5.9 pmol/liter) who were receiving nifedipine. Platelet [Ca2+]i was increased in patients with elevated PTH, compared with those in whom PTH was normal (138 +/- 16 vs. 83 +/- 7 nmol/liter, P < 0.01). A linear relation was observed between serum PTH and platelet [Ca2+]i in these patients (r = 0.818, P < 0.001). In contrast, platelet [Ca2+]i was not elevated (84 +/- 9 nmol/liter) in the patients with elevated PTH who were receiving nifedipine. A linear relation was also present between both serum PTH (r = 0.616, P < 0.001) and platelet [Ca2+]i (r = 0.576, P < 0.005) and mean blood pressure. Nine patients with hyperparathyroidism were restudied after treatment with the vitamin D analogue alfacalcidol. This resulted in significant decreases in serum PTH (P < 0.01), platelet [Ca2+]i (P < 0.02), and mean blood pressure (P < 0.05). These studies indicate that [Ca2+]i may be increased early in renal failure, and that this increase occurs in association with both hyperparathyroidism and hypertension. Furthermore, treatment of hyperparathyroidism with alfacalcidol may result in reductions in both [Ca2+]i and blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet loss during plasma exchange is unaffected by in-line filters.

15 patients underwent plasma exchange using a standard 170-microns in-line filter with or without an additional 40-microns microaggregate filter in the return circuit. The mean platelet count fall immediately after plasma exchange in 15 patients was (54 +/- 6) x 10(9)/l and 48 h later was (23 +/- 8) x 10(9)/l representing a mean total platelet loss immediately after plasma exchange of (253 +/- 31) x 10(9) with no difference in the 40-microns filtered procedures. The mean platelet loss in the discarded plasma was (60 +/- 8) x 10(9), and a mean of 52 x 10(9) platelets were recovered from machine harness washings. The platelet loss in the removed plasma and in the harness therefore accounted for only 42-44% of the total loss of platelets. The inclusion of 40-microns microaggregate filters did not reduce platelet loss, and it is therefore unlikely that the thrombocytopenia is induced by reinfused microaggregates. It is likely that platelets are activated in the machine and sequestered in the spleen.

Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.

Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.