Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins.

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.

Free-calcium and tension responses in single barnacle muscle fibres following the application of L-glutamate.

The effect of the putative transmitter, L-glutamate, on free intracellular Ca2+, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of L-glutamate (0.1--10 mM) resulted in a transient increase in free intracellular Ca2+, monitored by the Ca2+-activated protein aequorin. This increase in free intracellular Ca2+ was associated with membrane depolarization and force development, and was followed by a period of 'desensitization' in which the preparation was unresponsive to L-glutamate. This could be reversed by removing L-glutamate from the external saline. External application of a number of closely related compounds, including D-glutamate and L-aspartate, were ineffective for initiating the transient light response. The L-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to L-glutamate were not reduced in Mg-free medium. The fibre's response to 1mM L-glutamate was also inhibited by D-600 (10 micrometer) or by La3+ (1mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.

The effect of 5-hydroxytryptamine and other indole derivatives on the formation of adenosine 3',5'-cyclic monophosphate in pigeon erythrocytes.

1. The effect of insulin, acetylcholine, histamine, 5-hydroxytryptamine and prostaglandins E1, E2 and F2alpha on basal and adrenalin-stimulated cyclic AMP content in intact pigeon erythrocytes was investigated. 2. None of these compounds influenced basal cyclic AMP contest, and only 5-hydroxytryptamine antagonized the effect of adrenalin. The increase in cyclic AMP with 0.55 micronM adrenalin was inhibited by approx. 60% in the presence of 10 muM 5-hydroxytryptamine. The interaction between adrenalin and 5-hydroxytryptamine was competitive. 3. 5-Hydroxytryptamine did not affect the rate of degradation of cyclic AMP in intact cells, but did inhibit adrenalin-stimulated cyclic AMP formation in permeable or resealed cell "ghosts". 4. The effect of 5-hydroxytryptamine to inhibit cyclic AMP accumulation was not dependent on the presence of Ca2+, in either intact cells or "ghosts". 5. Various indole derivatives and other compounds were tested for their ability to inhibit the effect of adrenalin on cyclic AMP accumulation. Only those derivatives with a free amino group and net positive charge in the side chain were effective. 6. It was concluded that 5-hydroxytryptamine inhibits adrenalin-stimulated adenylate cyclase activity in pigeon erythrocytes, possibly by competing with adrenalin for binding to the beta-adrenergic receptor.

Stepwise removal of the C-terminal 12 amino acids of firefly luciferase results in graded loss of activity.

The C-terminus of the firefly luciferase (550 amino acids) was engineered using PCR followed by in vitro transcription-translation in order to investigate the role of the last 12 amino acids in the bioluminescence. Coding sequences were removed stepwise and the decapeptide MRSAMSGLHL, a putative AMP-activated protein kinase phosphorylation site, was used to replace the last 8-12 amino acids in order to test for amino acid specificity at the C-terminus. Removal of up to seven of the C-terminal amino acids resulted in no detectable loss of bioluminescent activity. However, the luciferase activity decreased stepwise from 50 to 0.1% when 8-12 amino acids were removed. Replacement of amino acids 539-550 and 543-550 by MRSAMSGLHL generated luciferases that retained 22 and 35% of catalytic activity respectively. These results have important implications for the further development of engineered luciferases as intracellular indicators and the understanding of the active centre of beetle luciferases.

Preparation and characterization of liposomes containing the Ca2+-activated photoprotein, obelin.

1. Liposomes bearing different net surface charges have been prepared and their ability to entrap the Ca2+-activated photoprotein, obelin, has been studied 2. Negatively-charged liposomes, composed of egg-yolk lecithin, cholesterol and phosphatidylserine, consistently produced the most homogeneous populations of liposomes after sonication, as shown by electron microscopy after negative staining. These consisted of a large proportion of uni- and bilamellar vesicles within the size range of 20--50 nm, external diameter. 3. Sonicated negatively-charged liposomes had a mean aqueous obelin space of 6.8 +/- 0.8 microliter/mumol of phospholipid compared to a mean space for inulin [14C]carboxylic acid of 5.1 +/- 0.8 microliter/mumol of phospholipid. 4. Sonication reduced the Ca2+ permeability of the negatively-charged liposomes, as measured by the utilization of entrapped obelin. 5. Preparations of uncharged (no phosphatidylserine) and positively-charged (stearylamine instead of phosphatidylserine), sonicated liposomes contained a greater proportion of larger vesicles, which were more permeable to Ca2+ than sonicated, negatively-charged liposomes. 6. Obelin, trapped within sonicated, negatively-charged liposomes, responded to increases in the free Ca2+ concentration within the liposomes caused by the bivalent-cation ionophore A23187 at concentrations as low as 19 nM. 7. The effect of A23187 was inhibited by Mg2+ at a low concentration of Ca2+ (10 muM), but not at 1 mM Ca2+. 8. It was concluded that obelin could be trapped in the aqueous compartment of sonicated liposomes which remained relatively impermeable to Ca2+. Furthermore, trapped obelin could respond to changes in the free Ca2+ concentration within these liposomes.

Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin.

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin. 2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca-2+ for aequorin and at least 3Ca-2+ for obelin. 3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units. 4. Other monovalent cations, such as Na+ are able to compete with Ca-2+ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions. 5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca-2+ solutions or of calcium/EGTA buffers.

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins.

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06-120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in development of bioluminescent indicators of the covalent modification of proteins.

Low erythrocyte folate, but not plasma vitamin B-12 or homocysteine,is associated with dementia in elderly Latinos.

The relationship between B vitamin status and cognitive function has been of interest for many years. There is evidence of relationships between intake and status of folate and vitamin B-12 with neurological, cognitive, and memory impairment, but results have been inconsistent. Plasma B-12, erythrocyte folate, methylmalonic acid,and homocysteine were evaluated as predictors of cognitive function in a large population based sample of Latino elderly living in the Sacramento, California region. The hypothesis tested was that low folate and/or B-12 status predicts cognitive function impairment and dementia. Logistic regression was used to examine the differences in B-vitamin status by cognitive function category. Erythrocyte folate was related to dementia after controlling for age, gender, education, income, diabetes diagnosis, serum creatinine, and depressive symptoms. The highest prevalence of low erythrocyte folate occurred in the Dementia group and was significantly higher than in the Normal group. Plasma B-12, MMA, Hcy, and prevalence of a normal values for these variables, were not significantly different among the cognitive function classes. We conclude that folate status is associated with dementia but that more research is needed on the relationship between vitaminB-12 status, Hcy and cognitive function to explore possible associations with these parameters.

Systemic lactose intolerance: a new perspective on an old problem.

Intolerance to certain foods can cause a range of gut and systemic symptoms. The possibility that these can be caused by lactose has been missed because of "hidden" lactose added to many foods and drinks inadequately labelled, confusing diagnosis based on dietary removal of dairy foods. Two polymorphisms, C/T13910 and G/A22018, linked to hypolactasia, correlate with breath hydrogen and symptoms after lactose. This, with a 48 hour record of gut and systemic symptoms and a six hour breath hydrogen test, provides a new approach to the clinical management of lactose intolerance. The key is the prolonged effect of dietary removal of lactose. Patients diagnosed as lactose intolerant must be advised of "risk" foods, inadequately labelled, including processed meats, bread, cake mixes, soft drinks, and lagers. This review highlights the wide range of systemic symptoms caused by lactose intolerance. This has important implications for the management of irritable bowel syndrome, and for doctors of many specialties.

A mechanism for the insertion of complement component C9 into target membranes.

Complement component C9 is a globular serum protein which can insert and polymerise in a target membrane to form a large membrane channel. The ability to insert in the membrane is conferred by amphipathetic elements of secondary structure in the central part of the molecule. Towards each end high cysteine domains are found, one of which is homologous to the apoprotein binding domains of the LDL receptor. From the sequence and topological data for C9 we present a model for its structure and insertion into the membrane.

Is intracellular Ca2+ the trigger for oxygen radical production by polymorphonuclear leucocytes?

The aim of this paper is critically to evaluate the existing evidence for the role of intracellular Ca2+ in polymorphonuclear leucocyte (PMN) activation and in particular in oxygen radical production. Indirect experiments are based on the manipulation of extracellular Ca2+, measurement of 45Ca fluxes, employing pharmacological agents such as Ca2+-ionophores and intracellular Ca2+ antagonists and monitoring chlortetracycline fluorescence. Experiments of this type do not provide the necessary definitive evidence that an increase in intracellular Ca2+ is the trigger for PMN activation. Recent direct measurements of intracellular free Ca2+ using the Ca2+-activated photoprotein, obelin, and the Ca2+-sensitive fluorescent indicator, quin 2, have provided evidence for the existence of two distinct mechanisms of activation, one triggered by a rise in intracellular Ca2+ and the other independent of a rise in intracellular Ca2+. The source of the Ca2+ for the former mechanism is mainly extracellular but can also come from an intracellular Ca2+ store.

Intracellular Ca2+ and cell injury: a paradoxical role of Ca2+ in complement membrane attack.

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.

Complement component C9-dependent cytosolic free Ca2+ rise and recovery in neutrophils.

The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.

Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector.

The physiologic relevance of Ca2+ release from the sarcoplasmic reticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmic reticulum free [Ca2+] (i.e. [Ca2+]sr) in the intact rat tail artery. We exploited a novel technique to measure [Ca2+]sr with genetically targeted apoaequorin acting as a pseudo-luciferase rather than as classic aequorin. Intact rat tail arteries were infected with a replication deficient adenoviral vector (RAdER) containing the apoaequorin gene targeted to the sarcoplasmic reticulum. Addition of apoaequorin's substrate, coelenterazine, to the perfusate increased light production in a [Ca2+] dependent manner, consistent with apoaequorin action on coelenterazine. Within the limits of the photon counting system, imaging of infected rat tail artery segments revealed light production from the whole thickness of the vascular wall. Phenylephrine stimulation decreased apoaequorin generated light and induced a contraction. Washout of phenylephrine relaxed the tissues and increased light indicating refilling of the sarcoplasmic reticulum with Ca2+. Incubation in 10 microM cyclopiazonic acid, a SERCA inhibitor, did not alter apoaequorin generated light or induce a contraction. In the presence of cyclopiazonic acid, phenylephrine contractions were enhanced and apoaequorin generated light decreased further than that observed in the absence of cyclopiazonic acid. Cyclopiazonic acid also prevented the increase in apoaequorin generated light upon washout of phenylephrine, consistent with its inhibition of sarcoplasmic reticulum refilling. These results suggest that light production from targeted apoaequorin, delivered by a replication deficient adenovirus, is a valid measure of changes in [Ca2+]sr in the intact arterial wall. There appeared to be a correlation between Ca2+ release and contraction in these lightly loaded arteries.

Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells.

The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating them into live cells without impairment of cell function. We have developed three methods for incorporating photoproteins into small cells (a) reversible cell swelling (b) membrane fusion and (c) intracellular release from pinocytotic vesicles. Formation of the membrane attack complex of complement (C5b6789), via a specific cell surface antibody to activate complement, causes a rapid increase in cytoplasmic Ca2+ detectable within 5-10 s. It provides a specific method for quantifying cytoplasmic photoprotein. As a result new insights into the role of intracellular Ca2+ in cell physiology and pathology have been established.

Calcium imaging shows differential sensitivity to cooling and communication in luminous transgenic plants.

Imaging of a recombinant bioluminescent Ca2+ indicator, aequorin, in an entire organism showed three novel features of Ca2+ signals in plants. First, cooling the plant from 25 degrees C to 2 degrees C demonstrated differential sensitivities between organs, the roots firing a Ca2+ signal at some 8-10 degrees C higher than the cotyledons. Secondly, prolonged cooling provoked Ca2+ oscillations, but only in the cotyledons. These oscillations occurred with a frequency of 100 s and damped down within 800 s. Thirdly, cooling the roots of mature plants triggered a Ca2+ signal in the leaves, as a result of organ-organ communication. However, warming and then recooling the roots did not generate a second Ca2+ signal in these leaves. This desensitisation was not due to down-regulation in the leaf since this was able to generate a Ca2+ signal of its own when cooled directly. Thus a combination of a recombinant bioluminescent indicator with photon counting imaging reveals startling new aspects of signalling in intact organs and whole organisms.

Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli.

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal.

Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that the heterogeneity and asynchrony of the oxidase response originates from variability in the timing and magnitude of the cytosolic free Ca2+ signal. The Ca2+ signals from individual cells could be classified into four types: (a) type 1, a transient rise in Ca2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free Ca2+; (c) type 3, a latent Ca2+ transient significantly delayed (21-56 s); and (d) type 4, no significant Ca2+ rise. These response types accounted for approximately 41%, 15%, 26% and 18% of the population respectively for stimulation with 1 microM f-met-leu-phe peptide (n = 27) and 52.5%, 15%, 11.5% and 21% respectively for 0.1 microM f-met-leu-phe peptide (n = 52). The oxidase in neutrophils in which the cytosolic free Ca2+ concentration rose to greater than 250 nM always became activated. In the presence of extracellular Ca2+, cytosolic Ca2+ rose uniformly throughout the cell, whereas in the absence of extracellular Ca2+, a localised Ca2+ 'cloud' was observed in approximately 30% of cells. A localised activation of the oxidase accompanied the presence of the Ca2+ 'cloud' when the 250 nM Ca2+ threshold was exceeded. The data presented here therefore demonstrate a tight coupling in individual neutrophils between an elevation in cytosolic free Ca2+ above a threshold of 250 nM and activation of the oxidase.

Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells.

Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].