Endoplasmic Reticulum Calcium ATPase Inhibitor Research Articles

Abstract 11427: Calcium Mobilization From Endoplasmic Reticulum Involves Calmodulin-mediated NADPH Oxidase-derived Reactive Oxygen Species Production in Porcine Aortic Endothelial Cells

Introduction: Previous studies indicate that calcium/calmodulin (Ca 2+ /CaM) mediates the phosphorylation and activation of NADPH oxidase (NOX). In endothelial cells, elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) level consists of two components, mobilization of Ca 2+ from intracellular stores and subsequent store-operated Ca 2+ entry (SOCE). However, little is known which component is required to regulate NOX-derived ROS production via Ca 2+ /CaM dependent pathway in endothelial cells. Hypothesis: We hypothesized that Ca 2+ mobilization from endoplasmic reticulum (ER), but not SOCE, is required to regulate NOX-derived ROS via Ca 2+ /CaM dependent pathway in porcine aortic endothelial cells (PAECs). Methods: We evaluated the association between Ca 2+ /CaM mediated NOX-derived ROS production and Ca 2+ mobilization from ER. We measured [Ca 2+ ] i by fura-2/AM a production of ROS by C-DCDHF-DA and in primary cultured PAECs with a fluorescence imaging and analysis system. Results: (1) In the presence of 1mM extracellular Ca 2+ ([Ca 2+ ] o ), BK induced a rapid increase [Ca 2+ ] i and followed by a sustained increase. However, in the absence of [Ca 2+ ] o (0mM with EGTA 1mM), BK caused only a small and transient increase [Ca 2+ ] i which was cause by Ca 2+ mobilization from ER. (2) BK (1μM) rapidly increased fluorescence intensity of C-DCDHF-DA compared with control. (150.2±51.3% and 107.2±5.6% of the baseline, respectively, p<0.05). (3) BK-induced ROS production was inhibited by an inhibitor of NOX (VAS2870: 50μM) (125.5±9.9% of the baseline, respectively, p<0.05). (4) When cells were exposed to BK with or without [Ca 2+ ] o , there was no difference in BK-induced ROS production. (5) In the absence of [Ca 2+ ] o , BK-induced ROS production is inhibited by an inhibitor of calmodulin (W-7: 100μM) (121.3±13.1% of the baseline, p<0.05). Thapsigargin (an inhibitor of ER calcium ATPase: 1μM) and BAPTA/AM (100μM) eliminated BK-induced ROS production (110.0±5.1% and 115.7±9.5% of the baseline, respectively, p<0.05 vs 1mM [Ca 2+ ] o ). Conclusions: The NOX-derived ROS production by BK is mediated via Ca 2+ /CaM dependent pathway. This was strictly regulated by Ca 2+ mobilization from ER.

Oleanolic acid induces relaxation and calcium‐independent release of endothelium‐derived nitric oxide

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO). Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs). Oleanolic acid (3-30 microM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 microM) and by the NO scavenger, oxyhaemoglobin (10 microM). Oleanolic acid-evoked NO increases were not reduced in Ca(2+)-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 microM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine(473) (Akt-Ser(473)) and eNOS at Serine(1177) (eNOS-Ser(1177)), which was attenuated by inhibitors of phosphoinositide-3-kinase. This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser(473) followed by phosphorylation of eNOS-Ser(1177).

Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: Potential role of protein phosphatase 2A–protein kinase CβII complex

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.

NO Calcium Signals from the Mitochondria to the ER

Xu et al. used human cell lines that expressed inducible nitric oxide synthase (iNOS) under the control of regulated promoters to investigate the effects of inhibiting mitochondrial respiration with nitric oxide (NO), which competes with oxygen for cytochrome c oxidase. NO, acting independently of soluble guanylate kinase activity, stimulated the expression of glucose-regulated protein 78 (Grp78), an endoplasmic reticulum (ER)-resident chaperone protein whose expression is enhanced as part of the ER stress response. The authors used Western analysis to show that NO produced an increase in the amount of the soluble transcription factor p50 ATF6, which has been implicated in the regulation of Grp78 expression. ATF6 is generated through a calcium-dependent process involving regulated intramembranous proteolysis. NO-dependent stimulation of p50 ATF6 production and of Grp78 expression was attenuated in cells depleted of intracellular calcium by exposure to a calcium ionophore in the absence of extracellular calcium. NO protected against cytotoxicity produced by the ER calcium ATPase inhibitor thapsigargin, and this protection (as well as the increase in Grp78 mRNA) was abrogated by silencing two serine proteases involved in the proteolytic generation of p50 ATF6 with siRNA. Both an intracellular calcium chelator and cyclosporin A (which interferes with mitochondrial calcium signaling) reduced NO-dependent ATF6 cleavage and prevented the NO-dependent increase in Grp78 and protection from thapsigargin cytotoxicity. Thus, the authors propose that NO-dependent inhibition of mitochondrial respiration affects calcium signaling between the mitochondria and the ER, thereby stimulating production of p50 ATF6 and thus of genes involved in the ER stress response. W. Xu, L. Liu, I. G. Charles, S. Moncada, Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. Nat. Cell Biol. 6 , 1129-1134 (2004). [Online Journal]

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.

EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca2+]i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca2+]i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 microM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca2+ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn2+ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 microM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 microM UO126 (MEK-1/2 inhibitor) or 10 microM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca2+]i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP3R isoforms 1-3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [3H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erkl/2 activity, whereas PKA stimulation suppresses these effects of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

17β-Estradiol and the phytoestrogen genistein attenuate neuronal apoptosis induced by the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin

Estrogenic compounds have been shown to protect neurons from a variety of toxic stimuli in vitro and in vivo and depletion of estrogen at menopause has been associated with increased risk of neurodegenerative diseases. Genistein is an isoflavone soy derivative that binds to estrogen receptors with selective estrogen receptor modulator (SERM) properties. Recent FDA recommendations of soy intake for cholesterol reduction have prompted investigation into the potentially estrogenic role of dietary soy phytochemicals in the brain. In this study, we have shown that 50 nM genistein significantly reduces neuronal apoptosis in an estrogen receptor-dependent manner. The importance of apoptosis in the brain has been recognized with regard to organization of the developing brain as well as degeneration in response to disease or stroke; however, the effects of estrogenic compounds on neuronal apoptosis have not been thoroughly examined. We developed a model of apoptotic toxicity in primary cortical neurons by using the endoplasmic reticulum (ER) calcium-ATPase inhibitor, thapsigargin, to test potential anti-apoptotic effects of 17β-estradiol and genistein. Estrogen receptor β, but not estrogen receptor α, was detected in our primary neuron cultures. Thapsigargin-induced apoptosis was confirmed by loss of mitochondrial function, DNA laddering, nuclear condensation and fragmentation, and caspase activation. Both 17β-estradiol and genistein reduced the number of apoptotic neurons and reduced the number of neurons containing active caspase-3. This effect was blocked by co-addition of ICI 182,780. Our results demonstrate that genistein and 17β-estradiol have comparable anti-apoptotic properties in primary cortical neurons and that these properties are mediated through estrogen receptors.

Effect of intracellular calcium modulation on sulfur mustard cytotoxicity in cultured human neonatal keratinocytes

Previous studies in human skin keratinocyte cultures have shown that sulfur mustard (HD) induces an immediate and irreversible increase in internal free calcium levels that was independent of external calcium concentrations. These findings suggested a role for calcium in the aetiology of HD-induced cell death and that modulation of intracellular calcium concentrations may assist in providing protection against this agent. In the current work, actively proliferating and confluent cultures of first passage neonatal human skin keratinocytes were used to assess the effect of altered intra- and extracellular calcium levels on HD toxicity. Treatment of cultures with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, or the calcium chelator BAPTA-AM, which reduce HD-induced elevation of intracellular free calcium, did not modulate the toxicity of HD. Furthermore, alteration of external calcium concentrations during these same experiments failed to elicit any change in the viability of HD-exposed cells. Treatment of confluent cultures with ionomycin at either low (100 μ m) or high (1.2 m m) external calcium concentrations also failed to modulate the toxicity of HD in any way. It appears that in neonatal human skin keratinocytes in culture, HD-induced intracellular calcium perturbation does not play a major role in HD-induced cytotoxicity.

Biochemical Requirements of Virus Wrapping by the Endoplasmic Reticulum: Involvement of ATP and Endoplasmic Reticulum Calcium Store during Envelopment of African Swine Fever Virus

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae.

Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle.

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle.

Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera.

Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+]i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+]i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+]i, they showed contrasting sensitivities depending on whether the [Ca2+]i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+]i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+]i at the plasma membrane as distinct from global changes in [Ca2+]i within the cytosol.

An increase in [Ca 2+ ] i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Elevates Basal B-Cell Intracellular Calcium Concentration and Suppresses Surface Ig- but Not CD40-Induced Antibody Secretion

Humoral immune responses to either T-independent or T-dependent antigens have previously been shown to be suppressed by the halogenated aromatic hydrocarbon environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through direct action on B-lymphocytes. To better understand the molecular nature of the TCDD-induced suppression of B-cell differentiation, we studied the effects of TCDD usingin vitromodels of T-independent (antibody directed against surface IgM) and T-dependent [activated T-helper (TH)-cells bearing CD40 ligand] B-cell maturation. We report here that TCDD suppresses murine B-cell IgM secretion induced by either soluble or insolubilized anti-IgM plus lymphokines but does not affect IgM secretion stimulated by activated TH-cells and lymphokines. Because soluble or insolubilized anti-IgM but not fixed, activated TH-cells was found to trigger increases in intracellular ionized calcium in isolated B-cells, the effect of TCDD exposure on B-cell intracellular calcium concentration and mobilization was examined. In comparison to the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, which induces an immediate rise in resting [Ca2+]iof up to four- to fivefold, TCDD treatment did not produce a rapid increase in [Ca2+]ibut did result in an elevation of basal levels of nearly the same magnitude 18 hr postexposure. However, anti-IgM-induced calcium transients were similar in the presence or absence of TCDD. TCDD exposure also produced instability of the calcium concentration curve, with the observed elevation of basal intracellular calcium occurring after bothin vitroandin vivotreatment paradigms. The immunomodulatory profiles of activity of TCDD and thapsigargin on the B-cell proliferative response to PMA plus ionomycin differ, suggesting that the kinetics of calcium release by these compounds dictates the overall effect on the responding B-cell. Taken together, the data indicate that TCDD elevates resting intracellular calcium levels in murine B-cells and may selectively inhibit calcium-dependent signaling pathways linked to surface Ig.

Blocking Ca 2+ mobilization with thapsigargin reduces neurite initiation in cultured adult rat DRG neurons

Adult rat DRG neurons rapidly extend extensive neuritic arbors after a 1–2-day delay in culture and generate large depolarization-induced calcium signals during this time period that are derived primarily from intracellular calcium release. To assess whether intracellular calcium mobilization is required for neurite initiation, calcium stores were depleted by brief exposure to the irreversible endoplasmic reticulum calcium ATPase inhibitor thapsigargin; cultures were then maintained for 3 days, immunostained for neurofilament and scored for percentage of neurons with neurites at least twice as long as the cell body. Brief thapsigargin treatment (20 min) during the first 24 h in culture resulted in a substantial decrease in neurite initiation frequency without affecting neuronal or nonneuronal cell survival, suggesting that intracellular calcium mobilization is necessary for triggering neurite initiation in these neurons.

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells.

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.