Transition from metaphase to anaphase is accompanied by local changes in cytoplasmic free calcium in Pt K2 kidney epithelial cells.

We have used a Ca2+-sensitive dye, fura-2, to investigate the role of Ca2+ during mitosis in Pt K2 epithelial cells. The concentration of cytoplasmic free calcium, [Ca2+]i, increased 2-fold between metaphase and anaphase. Digital image analysis revealed two patterns of [Ca2+]i localization during anaphase. In half of the anaphase cells, the increase in [Ca2+]i was greatest in the region near the spindle poles and decreased radially. In the other anaphase cells, there was a ring of high [Ca2+]i in the cytoplasm, surrounding an area of low [Ca2+]i in the spindle midzone. Although the reason for the different patterns is not known, peak [Ca2+]i in both cases was sufficient to maintain a 2- to 6-fold gradient in [Ca2+]i from the polar region to the midzone. [Ca2+]i gradients may thus regulate spindle microtubule equilibria and directed chromosome movement during mitosis.     

Intracellular free calcium in response to oxytocin in pig endometrial cells

Intracellular free calcium concentration ([Ca2+]i) in response to oxytocin (OT) was studied in stromal, glandular epithelial and luminal epithelial cells obtained from the endometrium of gilts 16 days post-estrus. The amplitude of increased [Ca2+]i in response to 100 nM OT was greatest in stromal cells, intermediate in glandular epithelial cells and not evident in luminal epithelial cells. During continuous OT administration, stromal cells responded initially with a synchronous spike of [Ca2+]i that did not require extracellular Ca2+ and then displayed spontaneous asynchronous [Ca2+]i spikes that required extracellular Ca2+. Each cell possessed its own characteristic response. Increasing concentrations of OT induced an increasing percentage of stromal cells responding, with some cells having nearly equal [Ca2+]i responses at all concentrations and others having graded [Ca2+]i responses as the concentration of OT increased. These results are consistent with the proposed mechanism of OT action in pig endometrium involving activation of phosphoinositide-Ca2+ signaling pathway.     

Clara cell protein-positive epithelial cells are reduced in small airways of asthmatics.

Clara cell 10 kilodalton protein (CC10), the predominant product from nonciliated cells in the epithelial lining of bronchioles (Clara cells), has been shown to have immunomodulatory and anti-inflammatory activity, and may play roles in controlling inflammation in the airway. This study was designed to examine immunohistochemical expression of CC10 in epithelial cells in small airways (perimeter < 6 mm) of asthmatic and control nonsmokers who underwent lung resection because of peripheral lung carcinoma and to compare CC10-positive epithelial cell proportions with numbers of inflammatory cells in small airways of asthmatics. Significantly decreased proportions of CC10-positive epithelial cells and significantly increased numbers of T cells, activated eosinophils, and mast cells in small airways of asthmatics were found compared with those of control subjects. CC10-positive epithelial cell proportions inversely correlated with numbers of T cells and mast cells in small airways of asthmatics. Decreases of CC10-producing cells may give an accelerating cause for further aggravation of inflammatory responses in chronic asthma.     

Intracellular polyamine levels of intestinal epithelial cells in inflammatory bowel disease

Polyamines and their acetylated derivatives are a prerequisite for cellular metabolism and considered to be essential for proliferation and differentiation of the rapidly renewing intestinal mucosa. However, their role during mucosal inflammation is less clear. Polyamine concentrations were determined in isolated colonic epithelial cells (CECs) from endoscopic biopsies from 26 patients with inflammatory bowel disease (IBD) and 40 controls as well as colon samples from mice with and without acute or chronic dextran sodium sulfate (DSS)-induced colitis. In patients with ulcerative colitis, CEC spermidine and N8-acetylspermidine levels were significantly enhanced and spermine levels were reduced compared with healthy controls. A correlation of polyamine levels of patients with IBD with their corresponding inflammatory index revealed that increased concentrations of spermidine, N8-acetylspermidine, and N1-acetylspermine were found in CECs from the most severe inflamed mucosal areas. Using acute and chronic DSS colitis as a model of mucosal inflammation, we found enhanced levels of spermidine and spermine in acute colitis, whereas in chronic inflammation, CEC spermine concentrations were decreased. Our data indicate a lack of the anti-inflammatory polyamine spermine in severe ulcerative colitis and chronic DSS colitis, which may aggravate the disease. Increased spermidine and N8-acetylspermidine levels reflect increased uptake and metabolism likely due to accelerated proliferation and regeneration of CECs.     

Cytosolic free calcium levels in sickle red blood cells

In this study, we used a recently developed nuclear magnetic resonance (NMR) technique to measure ionized calcium in sickle erythrocytes. The NMR technique, which involves 19F NMR studies of a fluorinated calcium chelator quinMF, [2-(2-amino-4-methyl-5-fluorophenoxy)methyl-8- aminoquinoline-N,N,N',N'- tetraacetic acid] provides a novel approach to the study of ionized calcium in erythrocytes since the presence of hemoglobin precludes the use of fluorescent calcium indicators. The mean value for ionized calcium in oxygenated sickle erythrocytes was 18 +/- 2 nmol/L (SE). Experiments with normal RBCs gave a mean value of 21 +/- 2 nmol/L (SE). After 1 hour of deoxygenation, mean values for ionized calcium in sickle erythrocytes did not increase as compared with values obtained under oxygen. To investigate whether deoxygenation stimulated endocytosis, sickle erythrocytes were deoxygenated for 1 hour in the presence of impermeant FBAPTA (1,2 bis-(2-amino-5- fluorophenoxy) ethane N,N,N',N'-tetraacetic acid). Cells were then separated from the extracellular medium and assayed for the presence of FBAPTA; they had incorporated significant quantities of the extracellular FBAPTA. This incorporation was not observed with normal erythrocytes. These data are consistent with at least a portion of the elevation in total cell calcium in sickle erythrocytes arising as a consequence of an endocytotic process in which extracellular calcium ions are incorporated into vesicles. Additional experiments show that these intracellular vesicles accumulate Ca2+ on further deoxygenation, consistent with a transient increase in ionized cell calcium. These studies represent the first use of NMR spectroscopy to evaluate endocytotic processes.     

Intracellular free ionized calcium in the pathogenesis of acute pancreatitis.

Acute pancreatitis is a common, often severe disease with multiple causes. Many of the aetiological factors responsible for triggering acute pancreatitis have been identified but the pathophysiological mechanism by which they do so is still poorly understood. Free calcium ions within the cytosol of the acinar cell ([Ca2+]i) act as a key intracellular second messenger in the processes of stimulus-secretion coupling and may be crucial in the pathogenesis of acute pancreatitis. [Ca2+]i signals have been shown to be disrupted early in experimental pancreatitis, and it is known that an abnormal rise in [Ca2+]i is toxic by a variety of mechanisms. It has been demonstrated that abnormal, prolonged elevations in [Ca2+]i result from caerulein hyperstimulation and ethanol treatment, and it is likely that all the known causes of acute pancreatitis can cause similar disruptions. Elevations in [Ca2+]i have also been shown to be associated with both acinar cell vacuolization and intracellular enzyme activation, both of which are key steps in the pathogenesis of acute pancreatitis. A disturbance of intracellular Ca2+ signalling and the generation of an abnormal elevation in [Ca2+]i appears to be the common factor linking all the known triggers for acute pancreatitis and initiating the further sequence of pathological events leading to clinical disease.     

Nitric oxide donors modify free intracellular calcium levels in rat anterior pituitary cells

The effect of nitric oxide donors on intracellular calcium concentration [Ca2+]i was studied in anterior pituitary cells using ratiometric FURA 2 fluorescence measurements. Sodium nitroprusside (NP) induced a transient decrease in [Ca2+]i, after which [Ca2+]i returned to, or even increased over basal values. S-Nitroso glutathione (GSNO) induced a similar decrease. NP also inhibited high [Ca2+]i achieved by depolarization with 25 mM K+. The inhibitory effect of NP was partially blunted by pretreatment with methoxy-verapamil, and in calcium free buffer, and was not altered by thapsigargin. Interestingly, in calcium free buffer there was a significant stimulatory effect of NP, which was partially blunted by thapsigargin. We conclude that NO donors modify [Ca2+]i in anterior pituitary cells. The action is biphasic, with an initial decrease in [Ca2+]i probably related to a decrease of Ca2+ influx through VDCC, and an increase evidenced in calcium free buffer in which the inhibitory component is absent, and partially depends on thapsigargin sensitive calcium stores.     

Intracellular free calcium concentration measured with 19F NMR spectroscopy in intact ferret hearts.

Changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediate excitation-contraction coupling in the heart and contribute to cellular injury during ischemia and reperfusion. To study these processes directly, we measured [Ca2+]i in perfused ferret (Mustela putorius furo) hearts using 19F NMR spectroscopy to detect the 5,5'-difluoro derivative of the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). To load cells, hearts were perfused with the acetoxymethyl ester derivative of 5,5'-F2-BAPTA. We measured 19F NMR spectra and left ventricular pressure simultaneously, at rest and during pacing at various external Ca concentrations [( Ca]o). Although contractile force was attenuated by the Ca2+ buffering properties of 5,5'-F2-BAPTA, the decrease in pressure could be overcome by raising [Ca]o. Our mean value of 104 nM for [Ca2+]i at rest in the perfused heart agrees well with previous measurements in isolated ventricular muscle. During pacing at 0.6-4 Hz, time-averaged [Ca2+]i increased; the effect of pacing was augmented by increasing [Ca]o. [Ca2+]i more than tripled during 10-20 min of global ischemia, and returned toward control levels upon reperfusion. This approach promises to be particularly useful in investigating the physiology of intact hearts and the pathophysiology of alterations in the coronary circulation.     

Abnormal secretagogue-induced intracellular free Ca2+ regulation in cystic fibrosis nasal epithelial cells.

These studies identify a further abnormality in cystic fibrosis (CF). The increase in intracellular free calcium concentration ([Ca2+]i) after exposure to histamine and PGE1 is demonstrated to be abnormally low in nasal cells, studied in short-term culture, from patients with CF compared with control subjects. [Ca2+]i is measured by using the Ca(2+)-sensitive fluorescent dye fura-2 and a fluorescence microscope imaging system. The percentage of CF cells that increase [Ca2+]i in response to histamine is decreased compared with controls, and, even in those CF cells that increase [Ca2+]i, the magnitude of the increase in [Ca2+]i in response to histamine is smaller than in controls. When exposed to PGE1, a similar number of control and CF cells responded with an increase in [Ca2+]i, but again the magnitude of the response was smaller in the CF cells. The mechanism of the PGE1-induced increase in [Ca2+]i is not mediated by cAMP, since 8-bromo-cAMP failed to increase [Ca2+]i in these cells. This abnormality in [Ca2+]i response did not apply to all secretagogues, with the response to carbachol being similar in CF and normal cells. How the abnormal CF gene product accounts for the abnormality in intracellular Ca2+ response to some but not all secretagogues is unknown.     

Intracellular calcium transients and arrhythmia in isolated heart cells

Intracellular calcium ([Ca2+]i) elevation may mediate cardiac arrhythmias. However, direct measurement of the rapid alterations of [Ca2+]i on a beat-to-beat basis using fast temporal resolution and without signal averaging in the spontaneously beating in vivo heart is lacking. Furthermore, data from an isolated spontaneously beating myocyte preparation that develops arrhythmia similar to that in the in vivo heart are unavailable. We measured rapid changes of [Ca2+]i with fast temporal resolution in isolated spontaneously beating neonatal rat ventricular myocytes with cell-to-cell communication and characterized the interrelation between [Ca2+]i and arrhythmia. An elevated extracellular calcium ([Ca2+]o) concentration of 10.8 mM induced premature beats, a rapid beating rate (tachyarrhythmia), and chaotic or fibrillatory beating activity in a small group of myocytes. [Ca2+]i levels during systole increased from the nanomolar to micromolar concentration range before arrhythmia development. Spontaneous oscillations of [Ca2+]i during diastole could evoke a spontaneous tachyarrhythmia. In the presence of [Ca2+]i elevation, a spontaneous tachyarrhythmia could induce severe [Ca2+]i overload. Reduction of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid AM (5 microM) in the presence of 10.8 mM [Ca2+]o reversed the arrhythmia. In single ventricular myocytes superfused with 10.8 mM [Ca2+]o, oscillations of membrane potential characteristic of transient inward current occurred that were prevented by ryanodine (0.1 microM), an inhibitor of Ca2+ flux across the sarcoplasmic reticulum. This study characterizes 1) an isolated multicellular myocyte model of arrhythmia similar to that evident in in vivo hearts, 2) elevation of [Ca2+]i with systolic [Ca2+]i levels of 1-3 microM and diastolic [Ca2+]i oscillations before the initiation of arrhythmia, 3) tachyarrhythmia as a cause of severe [Ca2+]i overload, which may be important in the perpetuation and degeneration of arrhythmias, and 4) reversal of arrhythmia with reduction of [Ca2+]i. The results in the isolated myocyte model may have relevance to the generation and perpetuation of certain cardiac arrhythmias associated with calcium overload.     

Claudin-5 levels are reduced in human end-stage cardiomyopathy

Claudin-5 is a transmembrane cell junction protein that is a component of tight junctions in endothelial cell layers. We have previously shown that claudin-5 also localizes to lateral membranes of murine cardiomyocytes at their junction with the extracellular matrix. Claudin-5 levels are specifically reduced in myocytes from a mouse model of muscular dystrophy with cardiomyopathy. To establish whether claudin-5 is similarly specifically reduced in human cardiomyopathy, we compared the levels of claudin-5 with other cell junction proteins in 62 cardiomyopathic end-stage explant samples. We show that claudin-5 levels are reduced in at least 60% of patient samples compared with non-failing controls. Importantly, claudin-5 reductions can be independent of connexin-43, a gap junction protein previously reported to be reduced in failing heart samples. Other cell junction proteins including α-catenin, β-catenin, γ-catenin, desmoplakin, and N-cadherin are reduced in only a small number of failing samples and only in combination with reduced claudin-5 or connexin-43 levels. We also show that reduced claudin-5 levels can be present independently from dystrophin alterations, which are known to be capable of causing and resulting from cardiomyopathy. These data are the first to show alterations of a tight junction protein in human cardiomyopathy samples and suggest that claudin-5 may participate in novel mechanisms in the pathway to end-stage heart failure.     

Regulatory T cells and IL-10 levels are reduced in patients with vulnerable coronary plaques

BackgroundDespite having a similar large extent of atherosclerotic coronary affliction, some patients suffer of recurrent cardiac events, whereas others remain asymptomatic.HypothesisWe hypothesized the existence of a systemic “signature” that could distinguish “vulnerable” patients with preexisting coronary atherosclerosis from those having similar risk factors and atheromatous burden, but no history of clinically evident plaque rupture/erosion.MethodsTwenty three patients who had at least two prior myocardial infarctions (“vulnerable group”) were matched in respect to their background and coronary atherosclerosis extent with twenty one patients without a history of previous myocardial infarction who underwent routine coronary angiography before valvular surgery. We studied a panel of cytokines, antibodies and hormones including IL-6, IL-10, IL-12, antibodies to β2 glycoprotein I (β2GPI), antibodies to oxidized-LDL, adiponectin and resistin, along with levels of circulating EPCs and Tregs.ResultsA significantly higher level of Treg cells was present in the control (73.4% ± 4) than in the “vulnerable patient” group (62.2% ± 10.7), p < 0.001. IL-10 level was also significantly higher in the control than in the vulnerable patients (2.6 ± 1.2 pg/ml versus 0.9 ± 0.1 pg/ml respectively, p = 0.03). There was no significant difference in the circulating levels of the other cytokines, hormones or EPCs between the two groups.ConclusionRegulatory T cells and serum IL-10 may discriminate “vulnerable” versus stable patients and may have a protective role against plaque rupture in patients with coronary atherosclerosis.     

Decreases in Raf-1 levels in galactosaemic lens epithelial cells are partially reversed by myo-inositol

Changes in the amounts and localization of protein kinase C (PKC) isoforms occur in galactosaemic lens epithelial cells. A link between PKC changes and myo-inositol depletion has been suggested. Raf-1, a component of a Ras pathway, is a substrate for PKC. Raf-1 levels were measured in galactosaemic lens epithelial cells grown with or without myoinositol. Raf-1 levels were measured by densitometric scanning of Western blots from cells grown with or without 40 mmol/l galactose or 40 mmol/l galactose plus 1.0 μmol/l myoinositol for 1, 3, 5 or 7 days. Scans were compared to those for PKCα, an isoform of PKC and to 14-3-3, a protein which binds to Raf-1. Cell growth was quantitated by thymidine incorporation. Raf-1 levels were decreased in bovine lens epithelial cells after 3, 5 or 7 days (33% of control) of growth in 40 mmol/l galactose. Addition of 1 μmol/l myoinositol reversed this decrease at day 3, but not after 5 or 7 days of growth in 40 mmol/l galactose. PKCα and 14-3-3 levels were not affected by galactose. The decrease in Raf-1 was not a result of cell growth as measured by thymidine incorporation. These results suggest that Raf-1 levels are decreased during galactosaemia. This was only partially reversed by the addition of myoinositol. Received: 14 November 1997 / Accepted in revised form: 24 June 1998     

Carbachol increases intracellular free calcium concentrations in human granulosa-lutein cells.

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70-140 nmol/l and maximal, carbachol-induced peaks reached 1.1 mumol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells.     

Intracellular free calcium and mitochondrial membrane potential in ischemia/reperfusion and preconditioning

Moderation of calcium perturbations has been implicated in ischemic preconditioning. As mitochondria possess an effective Ca(2+)transporting system driven by the mitochondrial membrane potential, experiments were performed to study time-averaged intracellular free calcium and the mitochondrial membrane potential during preconditioning and ischemia-reperfusion. Isolated rat hearts were subjected to 5 min of preconditioning, a 9-min intervening reperfusion and 21 min of ischemia with subsequent reperfusion. The hearts were preloaded with the Ca(2+)indicator Fura-2 or the mitochondrial membrane potential probe safranine. A method was devised for correction for NADH autofluorescence in time-averaged Ca(2+)probing with Fura-2. The pH dependence of the apparent dissociation constant of the Ca(2+)complex of Fura-2 was determined. Intracellular free Ca(2+)increased during the 5-min ischemia, and this was reversed upon reperfusion. During protracted ischemia a continual Ca(2+)rise was observed when the fluorescence data were corrected for changes in pH. An initial sharp Fura-2 fluorescence spike upon final reperfusion was caused by a pH-dependent change in the dissociation constant of the Ca(2+)complex of Fura-2. In preconditioned hearts the free Ca(2+)was somewhat lower during reperfusion, but a major effect of preconditioning was observed during the prolonged ischemia. The decrease in mitochondrial membrane potential during prolonged ischemia was faster in the preconditioned heart with no difference during the final reperfusion. The effect of preconditioning on cell survival was reflected in a decrease in the post-ischemic washout of creatine kinase. The moderation of the ischemic and post-ischemic intracellular Ca(2+)increase, and the acceleration of the ischemic mitochondrial membrane potential decrease by ischemic preconditioning is in accord with the notion of the involvement of mitochondrial ATP sensitive K(+)channels in preconditioning. In studies on ischemia it is absolutely necessary to correct for the pH-sensitivity of the apparent dissociation constant of the calcium complex of Fura-2 to obtain reliable data for intracellular free calcium.     

Prostaglandin F2 alpha and gonadotropin-releasing hormone increase intracellular free calcium in rat granulosa cells.

Changes in cytosolic free calcium concentration ([Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) and gonadotropin-releasing hormone (GnRH) were measured in single rat granulosa cells, using the calcium-sensitive fluorescent dye, fura-2AM. In 90 out of 135 granulosa cells (67%), there was a 3- to 4-fold increase in resting [Ca2+]i within 30 s of administration of PGF2 alpha (10(-6) M). The resting [Ca2+]i returned to pre-stimulation levels in approximately 80 s. Granulosa cells were responsive to PGF2 alpha at concentrations ranging from 10(-7) M to 10(-4) M (n = 7). Within this range of concentrations, the magnitude of the calcium response did not differ. In another series of experiments, the majority (93%, n = 57) of the granulosa cells which responded to PGF2 alpha also responded to GnRH. Neither the magnitude of the [Ca2+]i response nor the time to response differed between PGF2 alpha and GnRH. Furthermore, simultaneous treatment of granulosa cells with both hormones did not generate a larger response than with either hormone alone. During perifusion with low calcium media, the characteristic [Ca2+]i response to PGF2 alpha decreased, and was eliminated within 10 min (n = 9). Similar observations were made in response to GnRH under these conditions. These data confirm that PGF2 alpha and GnRH stimulate a transient increase in [Ca2+]i in rat granulosa cells, the source of which may be shared intracellular stores.     

Bone marrow cyclooxygenase-2 levels are elevated in chronic-phase chronic myeloid leukaemia and are associated with reduced survival

Increased angiogenesis is important in the pathophysiology of haematological malignancies. Cyclooxygenase-2 (Cox-2) converts arachidonic acid to prostaglandins, which induce expression of angiogenic factors, including vascular endothelial growth factor (VEGF), basic-fibroblast growth factor, transforming growth factor-beta and interleukin 6. Cox-2 may also reduce apoptosis and reduce cellular attachment to the extracellular matrix (ECM). Increased bone marrow (BM) vascularity, increased BM cellular and plasma VEGF levels, and decreased progenitor adherence to BM ECM have all been observed in chronic myeloid leukaemia (CML). We investigated the prognostic significance of levels of Cox-2 in BM cells from patients with CML. Western blot and solid-phase radioimmunoassay (RIA) were used to measure Cox-2 BM levels in 149 patients with chronic phase CML (CP CML). Results were compared with those of normal controls. Expression of Cox-2 was significantly higher in CML than in normal controls (P < 0.0001). Increasing levels of Cox-2 were significantly associated with shorter survival (P = 0.0002, Cox proportional hazard model). A multivariate model based on Cox-2 and degree of splenomegaly was developed for survival in patients with early CP CML. Agents that inhibit Cox-2 activity merit investigation in patients with CP CML.     

Haemopoietic progenitor cells are reduced in aplastic anaemia

Summary We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double-colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS), AA CD34⁺ cells were reduced by 68% compared to normal. In addition, AA CD33⁺ cells and the three progenitor subsets (CD34⁺/CD33^–, CD34⁺/CD33⁺ and CD34^–/CD33⁺) were reduced by 44–80%. Our data lend further support for an early stem cell deficiency in AA.     

Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum.

The effects of extracellular phosphate and lanthanum on cytosolic free Ca2+ [( Ca2+]i) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca2+ and 1.0 mM phosphate, the mean resting [Ca2+]i level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both [Ca2+]i levels and 45Ca2+ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca2+ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated [Ca2+]i levels were very rapid and were mimicked only by prolonged incubation of acini in Ca2+-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal [Ca2+]i levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting [Ca2+]i levels in pancreatic acini and other cell types and that mobilization of intracellular Ca2+ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca2+ that is not readily removed by EGTA.