Mechanisms of ATP-induced calcium signaling and growth arrest in human prostate cancer cells

This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.     

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha

Variations in calcium concentration within the endoplasmic reticulum ([Ca(2+)](ER)) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor-alpha (TNFalpha) as well as evaluating the possible role of [Ca(2+)](ER) variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca(2+)](ER) whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca(2+)](ER). IGF-induced [Ca(2+)](ER) increase is correlated to an overexpression of the sarcoendoplasmic calcium-ATPase 2B (SERCA2b), whereas TNFalpha-induced [Ca(2+)](ER) decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca(2+)](ER) reduction and SERCA2b downregulation. Reduction in [Ca(2+)](ER) induced by thapsigargin (TG) (from 1 pM to 1 microM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 microM TG. We also show that a transient TG application (1 pM, 1 nM, 1 microM 15 min) is insufficient to induce a long lasting decrease in [Ca(2+)](ER), since [Ca(2+)](ER) remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 microM, 15 min) induces apoptosis. We thus identify [Ca(2+)](ER) and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.     

Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.     

Caffeine releases a glucose-primed endoplasmic reticulum Ca2+ pool in the insulin secreting cell line INS-1

Caffeine mobilized an intracellular Ca2+ pool in intact fura-2-loaded INS-1 cells in suspension exposed to high (16 mM) [glucose], while a minor effect was observed with low (2 mM) [glucose]. Cells were kept in a medium containing diaxozide or no Ca2+ to prevent the influx of extracellular Ca2+. The caffeine-sensitive intracellular Ca2+ pool was within the endoplasmic reticulum since it was depleted by the inhibitor of the reticular Ca2+ pumps thapsigargin and the InsP3-dependent agonist carbachol. No effect of caffeine was observed in the parent glucose-insensitive RINmF5 cells. In microsomes from INS-1 but not RINmF5 cells, the type 2 ryanodine receptor was present as revealed by Western blotting. It was concluded that the endoplasmic reticulum of INS-1 cells possesses caffeine-sensitive type 2 ryanodine receptors Ca2+ channels.     

Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate‐derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR‐associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH‐mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells. J. Cell. Biochem. 114: 1322–1335, 2013. © 2012 Wiley Periodicals, Inc.     

Ca2+ homeostasis in apoptotic resistance of prostate cancer cells

Ca2+ is a universal messenger regulating many physiological functions including such an important one, as the ability of the cell to undergo orderly self-destruction upon completion of its mission, called apoptosis. If this function is compromised unwanted cells may eventually take over the tissue turning it into a cancer. Ca2+ dependency of apoptosis, when its all aspects are learned and understood and key molecular players identified, may provide a good opportunity for controlling tumor growth. In the present mini-review we describe the major molecular determinants of Ca2+ homeostasis in prostate cancer cells and establish their role in the transformation to apoptosis-resistant cell phenotypes typical of advanced androgen-independent prostate cancer. We show that the hallmark of such transformation is the inhibition of apoptosis pathway associated with endoplasmic reticulum Ca2+ store depletion.     

Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.     

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.     

Expression of endomembrane calcium pumps in colon and gastric cancer cells. Induction of SERCA3 expression during differentiation

Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.     

The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis

Enteroviruses, small cytolytic RNA viruses, confer an antiapoptotic state to infected cells in order to suppress infection-limiting apoptotic host cell responses. This antiapoptotic state also lends protection against cell death induced by metabolic inhibitors like actinomycin D and cycloheximide. The identity of the viral antiapoptotic protein and the underlying mechanism are unknown. Here, we provide evidence that the coxsackievirus 2B protein modulates apoptosis by manipulating intracellular Ca(2+) homeostasis. Using fluorescent Ca(2+) indicators and organelle-targeted aequorins, we demonstrate that ectopic expression of 2B in HeLa cells decreases the Ca(2+) content of both the endoplasmic reticulum and the Golgi, resulting in down-regulation of Ca(2+) signaling between these stores and the mitochondria, and increases the influx of extracellular Ca(2+). In our studies of the physiological importance of the 2B-induced alterations in Ca(2+) signaling, we found that the expression of 2B suppressed caspase activation and apoptotic cell death induced by various stimuli, including actinomycin D and cycloheximide. Mutants of 2B that were defective in reducing the Ca(2+) content of the stores failed to suppress apoptosis. These data implicate a functional role of the perturbation of intracellular Ca(2+) compartmentalization in the enteroviral strategy to suppress intrinsic apoptotic host cell responses. The putative down-regulation of an endoplasmic reticulum-dependent apoptotic pathway is discussed.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

The creatine kinase system is essential for optimal refill of the sarcoplasmic reticulum Ca2+ store in skeletal muscle.

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.     

Regulation of cell pH by Ca+2-mediated exocytotic insertion of H+- ATPases

Exposure to CO2 acidifies the cytosol of mitochondria-rich cells in turtle bladder epithelium. The result of the decrease in pH in these, the acid-secreting cells of the epithelium, is a transient increase in cell calcium, which causes exocytosis of vesicles containing proton-translocating ATPase. Because mitochondria-rich cells have rapid luminal membrane turnover, we were able to identify single mitochondria-rich cells by their endocytosis of rhodamine-tagged albumin. Using fluorescence emission of 5,6-carboxyfluorescein at two excitation wavelengths, we measured cell pH in these identified mitochondria-rich cells and found that although the cell pH fell, it recovered within 5 min despite continuous exposure to CO2. This pH recovery also occurred at the same rate in Na+-free media. However, pH recovery did not occur when luminal pH was 5.5, a condition under which the H+-pump does not function, suggesting that recovery of cell pH is due to the luminally located H+ ATPase. Chelation of extracellular calcium by EGTA prevented the CO2-induced rise in cell calcium measured with the intracellular fluorescent dyes Quin 2 or Fura 2 and also prevented recovery of cell pH. When the change in cell calcium was buffered by loading the cells with high concentrations of Quin 2, the CO2-induced decrease in pH did not return back to basal levels. We had found previously that buffering intracellular calcium transients prevented CO2-stimulated exocytosis. Further, we show here that the increased H+ current in voltage-clamped turtle bladders, which is directly proportional to the number of H+-pump-containing vesicles that fuse with the luminal membrane, was significantly reduced in calcium-depleted bladders. These results suggest that pH regulation in these acid-secreting cells occurs by calcium-dependent exocytosis of vesicles containing proton pumps, whose subsequent turnover restores the cell pH to its initial levels.     

Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.     

Selective Modulation of Endoplasmic Reticulum Stress Markers in Prostate Cancer Cells by a Standardized Mangosteen Fruit Extract

The increased proliferation of cancer cells is directly dependent on the increased activity of the endoplasmic reticulum (ER) machinery which is responsible for protein folding, assembly, and transport. In fact, it is so critical that perturbations in the endoplasmic reticulum can lead to apoptosis. This carefully regulated organelle represents a unique target of cancer cells while sparing healthy cells. In this study, a standardized mangosteen fruit extract (MFE) was evaluated for modulating ER stress proteins in prostate cancer. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells (PrECs) procured from two patients undergoing radical prostatectomy were treated with MFE. Flow cytometry, MTT, BrdU and Western blot were used to evaluate cell apoptosis, viability, proliferation and ER stress. Next, we evaluated MFE for microsomal stability and anti-cancer activity in nude mice. MFE induced apoptosis, decreased viability and proliferation in prostate cancer cells. MFE increased the expression of ER stress proteins. Interestingly, MFE selectively promotes ER stress in prostate cancer cells while sparing PrECs. MFE suppressed tumor growth in a xenograft tumor model without obvious toxicity. Mangosteen fruit extract selectively promotes endoplasmic reticulum stress in cancer cells while sparing non-tumorigenic prostate epithelial cells. Furthermore, in an in vivo setting mangosteen fruit extract significantly reduces xenograft tumor formation.     

Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.     

Simultaneous measurement of intracellular Ca(2+) and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have been established. We are going to present a new method to measure intracellular calcium and NO simultaneously in endothelial cells. A new fluorescent dye (DAF-2) has been developed recently which binds NO resulting in an enhanced fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 simultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin induces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This new method of a simultaneous measurement of intracellular calcium and NO provides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NO-synthase.