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• W2079740701 abstract "Endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through itsN-myristoylation, the significance of membrane association in locating eNOS near sources of Ca2+ entry is uncertain. To assess the Ca2+ source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca2+ sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca2+ concentration, demonstrating preferential sensitivity to Ca2+ originating from intracellular pools (MHA) or from capacitative Ca2+ entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca2+ influx via capacitative Ca2+ entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca2+release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca2+]i due to the Ca2+ ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca2+influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells. Endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through itsN-myristoylation, the significance of membrane association in locating eNOS near sources of Ca2+ entry is uncertain. To assess the Ca2+ source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca2+ sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca2+ concentration, demonstrating preferential sensitivity to Ca2+ originating from intracellular pools (MHA) or from capacitative Ca2+ entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca2+ influx via capacitative Ca2+ entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca2+release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca2+]i due to the Ca2+ ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca2+influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells. nitric oxide endothelial nitric-oxide synthase polymerase chain reaction intracellular Ca2 thapsigargin pulmonary artery endothelial cells capacitative Ca2+ entry eNOS-hemagglutinin-aequorin myristoylation-deficient EHA myristoylation-deficient eNOS extracellular Ca2+ concentration base pair(s) adenylyl cyclase type VI hemagglutinin base pair(s) 4,5-diaminofluorescence Nitric oxide (NO)1 is an ubiquitous intracellular signaling molecule, synthesized froml-arginine by nitric-oxide synthase (NOS) in diverse cells and tissues. There are three isoforms of NOS, first identified in neural tissue (nNOS, NOS 1) (1.Bredt D.S. Hwang P.W. Snyder S.H. Nature. 1990; 347: 768-770Crossref PubMed Scopus (2698) Google Scholar, 2.Bredt D.S. Hwang P.W. Glatt C.E. Lowenstein C.L. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar), endothelial cells (eNOS, NOS 3) (3.Pollock J.S. Förstermann U. Mitchell J.A. Warner T.D. Schmidt H.H.H.W. Nakane M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10480-10484Crossref PubMed Scopus (900) Google Scholar,4.Janssens S.P. Shimouchi A. Quertermous T. Bloch D. Bloch K.D. J. Biol. Chem. 1992; 267: 14519-14522Abstract Full Text PDF PubMed Google Scholar), and activated macrophages and hepatocytes (iNOS, NOS 2) (5.Stuehr D.J. Marletta M.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7738-7742Crossref PubMed Scopus (1019) Google Scholar, 6.Wright K.S. Mulsch A. Busse R. Osswald H. Biochem. Biophys. Res. Commun. 1989; 160: 813-819Crossref PubMed Scopus (273) Google Scholar), respectively. eNOS plays an major role in the control of blood pressure and vascular homeostasis. In the vascular endothelium, production of NO results in vascular smooth muscle relaxation which, in turn, reduces blood pressure. As expected, genetic ablation of the eNOS gene in mice results in systemic and pulmonary hypertension (7.Huang P.L. Huang Z. Moshimo H. Bloch K.D. Moskowitz M.A. Bevan J.S. Fishman M.C. Nature. 1995; 377: 239-242Crossref PubMed Scopus (1789) Google Scholar, 8.Shesely E.G. Maeda N. Kim H.-S. Desai K.M. Krege J.H. Laubach V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (792) Google Scholar). eNOS, a Ca2+/calmodulin-dependent enzyme, is highly regulated by intracellular Ca2+. Activation of eNOS is induced by increases in intracellular Ca2+ resulting from the activation of diverse G-protein-coupled cell surface receptors or from mobilization of intracellular Ca2+ stores. Previous studies have shown that thapsigargin (TG), a selective inhibitor of the Ca2+-ATPase on endoplasmic reticulum and sacroplasmic reticulum, activates NO release in pulmonary artery endothelial cells (PAEC) (9.Demura Y. Ishizaki T. Ameshima S. Okamura S. Hayashi T. Matsukawa S. Miyamori I. Br. J. Pharmacol. 1998; 125: 1180-1187Crossref PubMed Scopus (20) Google Scholar, 10.Wang Y. Shin W.S. Kawaguchi H. Inukai M. Kato M. Sakamoto A. Uehara Y. Miyamoto M. Shimamoto N. Korenaga R. Ando J. Toyo-oka T. J. Biol. Chem. 1996; 271: 5647-5655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, little is known about whether this activation results from intracellular Ca2+ release or from store-operated or capacitative Ca2+ entry (CCE) subsequent to depletion of the intracellular Ca2+ pool. eNOS is predominantly localized to caveolae, a specialized microdomain of the plasma membrane, which serves to compartmentalize signal transduction molecules (3.Pollock J.S. Förstermann U. Mitchell J.A. Warner T.D. Schmidt H.H.H.W. Nakane M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10480-10484Crossref PubMed Scopus (900) Google Scholar, 11.Förstermann U. Pollock J.S. Schmidt H.H.H.W. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar, 12.Hecker M. Mulsch A. Bassenge E. Förstermann U. Busse R. Biochem. J. 1994; 299: 247-252Crossref PubMed Scopus (120) Google Scholar). The caveolaen-eNOS interaction serves both to partition eNOS in caveolae and inhibit eNOS enzymatic activity. After Ca2+-calmodulin-dependent phosphorylation, eNOS becomes dissociated from caveolaen and becomes enzymatically activated. We hypothesize that an additional function of eNOS binding to caveolaen is to localize the enzyme in close proximity to CCE channels on the plasma membrane, and that the actual intracellular Ca2+that is required to activate this enzyme may be significantly different from that released from subcellular compartments as well as average [Ca2+]i. To test the hypothesis that CCE is essential for sustained activation of eNOS, EHA (eNOS-HA-aequorin) and MHA (myristoylation-deficient EHA) chimeras were generated and expressed in COS-7 and bovine PAEC. The EHA construct retained the ability to bind to caveolaen and target the plasma membrane, whereas the MHA construct was localized to the cytosol, and not to caveolae. Both constructs retained NOS and aequorin activity when expressed in COS-7 and PAEC. Using these two constructs, we investigated the relative contributions of release of intracellular Ca2+ versus CCE to the regulation of [Ca2+] in the region of eNOS and activation of NOS enzymatic activity under a variety of physiological conditions. We also measured NOS activation directly with an NO-sensitive fluorophore, comparing the ability to stimulate NO production of CCE induced by thapsigargin to that of generalized increase in [Ca2+]i produced by ionomycin. All procedures involving oligonucleotide and cDNA manipulations were performed essentially as described by Sambrook et al. (13.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Wild type eNOS (14.Lamas S. Marsden P.A. Li G.K. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Crossref PubMed Scopus (921) Google Scholar) and myristoylation-deficient (myr−) eNOS (15.Busconi L. Michel T. J. Biol. Chem. 1993; 268: 8410-8413Abstract Full Text PDF PubMed Google Scholar) cDNA in pBK-CMV vector (Stratagene, La Jolla, CA) were kindly provided by Dr. Thomas Michel (Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA). eNOS and ACVI/HA/AEQ containing adenylyl cyclase type VI (ACVI) and hemagglutinin (HA) epitope-tagged cytosolic aequorin (16.Nakahashi Y. Nelson E. Fagan K. Gonzales E. Guillou J.-L. Cooper D.M.F. J. Biol. Chem. 1997; 272: 18093-18097Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) were used as templates for polymerase chain reaction (PCR) amplification. To generate the eNOS-aequorin chimeric constructs, overlapping PCR was employed using a four-primer procedure (17.van den Hazel H.B. Kielland-Brandt M.C. Winther J.R. J. Biol. Chem. 1993; 268: 18002-18007Abstract Full Text PDF PubMed Google Scholar). The sequence of the oligonucleotide primers were as follows: primer 1, 5′-primer (5′-CCGCTCGAGCGGGGCCACATG-3′) specific to eNOS including the XhoI restriction site (underlined) located at nucleotide 3341 of the bovine eNOS cDNA; primer 2, 3′-primer (5′-ataatcaggaacatcataGGGGCCGGGGGTGTCTGG-3′) specific to the last 18 bp of eNOS coding sequence (uppercase letters) and first 18 bp of HA epitope tag (YDVPDYASL) of ACVI/HA/AEQ (lowercase letters); primer 3, 5′-primer (5′-CCAGACACCCCCGGCCCCtatgatgttcctgattat-3′) specific to 18 bp of eNOS (uppercase letters) and HA tag (lowercase letters), respectively; primer 4, 3′-primer (5′-CCTCTAGATTAGGGGACAGCTCCACC-3′) specific to the last 15 bp of cytosolic aequorin followed by a stop codon and XbaI site (underlined). The products of PCR1 (generated by primers 1 and 2) and PCR2 (generated by primers 3 and 4) were mixed in equimolar amounts, joined by the 36 bp of overlapping sequence (generated by the internal primers 2 and 3) and filled in by plaque forming unit polymerase (Stratagene); the combined product was amplified using the external primers 1 and 4 to generate the 900-bp PCR3 product. This product was subsequently digested with XhoI andXbaI restriction enzymes (Stratagene). To generate EHA construct, eNOS plasmid was digested with XhoI andXbaI to release 300 bp of the eNOS fragment which was replaced with the 900-bp XhoI/XbaI fragment of PCR3 product. The resulting constructs were sequenced in both directions through the entire sequence generated by PCR; no mutations were detected. Similar to the EHA construct, the MHA construct was generated by replacing the 300-bp XhoI/XbaI fragment of myr−-eNOS plasmid with the 900-bpXhoI/XbaI fragment from the EHA construct. The first 400 bp of both constructs were sequenced to confirm the point mutations resulting in glycine to alanine substitution at amino acid position 2 of the eNOS sequence in MHA construct. Both expression plasmids were transiently transfected into COS-7 cells and PAEC. The recombinant proteins were identified by immunoblotting with eNOS (Transduction Laboratories, Lexington, KY) or HA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antisera. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 at 37 °C. Bovine PAEC were isolated as described (18.Voyta J.C. Via C.P. Butterfield E.E. Zetter B.R. J. Cell Biol. 1989; 99: 2034-2040Crossref Scopus (1033) Google Scholar) and grown in minimal essential medium (Sigma) supplemented with 20% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO2 at 37 °C; PAEC used in this study were between passages 3 and 6. EHA and MHA expression plasmids were transiently transfected into PAEC and COS-7 cells using LipofectAMINE as described by the supplier (Life Technologies, Inc., Grand Island, NY). The transfection efficiency and eNOS expression were assessed by NADPH-diaphorase staining (19.Scherer-Singler U. Vincent S.R. Kimura H. McGeer E.G. J. Neurosci. Methods. 1983; 9: 229-234Crossref PubMed Scopus (683) Google Scholar, 20.Dawson T.M. Bredt D.S. Fotuhi M. Hwang P.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7797-7801Crossref PubMed Scopus (1841) Google Scholar). Experiments were performed 48 h after transfection. To determine the intracellular localization of EHA and MHA chimera, NOS enzymatic activity was assessed in subcellular fractions of COS-7 cells transfected with EHA or MHA constructs in the presence of excess substrates and cofactors as described previously (21.Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y.-S. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 22.Lantin-Hermoso R.L. Rosenfeld C.R. Yuhanna I.S. German Z. Chen Z. Shaul P.W. Am. J. Physiol. 1997; 273: L119-L126PubMed Google Scholar). Briefly, 48 h after transfection, cells were incubated with 0.75 μCi/ml [3H]l-arginine (Amersham Pharmacia Biotech) in the absence (basal) or presence of 100 nm TG and extracellular Ca2+ at 37 °C for 30 min followed by addition of 1 m trichloroacetic acid, and then lysed by freeze-thaw in liquid nitrogen. The samples were extracted three times with ether and passed through a Dowex AG50W X-8 column (Sigma). The [3H]l-citrulline generated was collected and quantified by liquid scintillation spectroscopy. Three independent experiments were done and results are expressed as the percentage of basal NOS activity in the same plate. The measurements of NOS enzymatic activity in the presence of excess substrates and cofactors reveal total NOS abundance in subcellular compartments. In vitro [Ca2+] calibration curves were determined using the membrane fraction of EHA-transfected cells or cytosolic fraction of MHA-transfected cells as described (16.Nakahashi Y. Nelson E. Fagan K. Gonzales E. Guillou J.-L. Cooper D.M.F. J. Biol. Chem. 1997; 272: 18093-18097Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,23.Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Cells were lysed and cytosolic fraction was separated from membrane fraction by centrifugation. Aequorin was reconstituted with 5 μm coelenterazine (Molecular Probes, Inc., Eugene, OR) in the presence of 140 mm β-mercaptoethanol for 3 h at 4 °C. Aequorin-mediated luminescence was measured with the LS50B Luminescence Spectrophotometer (Perkin-Elmer, Beaconsfield, United Kingdom) in the presence of various amounts of free Ca2+. The L max was obtained by integrating a continuous recording of aequorin-mediated light emission in the presence of 10 mm Ca2+. In vivo measurements of the effect of TG on aequorin in transfected COS-7 and endothelial cells were performed as described previously (16.Nakahashi Y. Nelson E. Fagan K. Gonzales E. Guillou J.-L. Cooper D.M.F. J. Biol. Chem. 1997; 272: 18093-18097Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cells were loaded with 5 μmcoelenterazine, washed, and resuspended in nominally Ca2+-free Krebs buffer (120 mm NaCl, 4.75 mm KCl, 1.44 mm MgSO4, 11 mm glucose, 25 mm HEPES, and 0.1% bovine serum albumin, pH 7.4) supplemented with 75 μm Ca2+and 0.2 mm EGTA was added immediately prior to measurements. 4 × 106 cells were used for each measurement. Luminescence was measured with the LS50B spectrophotometer and subsequently transformed into [Ca2+]i values as described by Brini et al. (23.Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar) and Allen et al. (24.Allen D.G. Blinks J.R. Prendergast F.G. Science. 1977; 195: 996-998Crossref PubMed Scopus (251) Google Scholar). For studies of the effect of histamine, luminiscence measurements were done exactly as described previously by Mersaultet al. (25.Marsault R. Murgia M. Pozzan T. Rizzuto R. EMBO J. 1997; 16: 1575-1581Crossref PubMed Scopus (163) Google Scholar) using a perfusion system. Briefly, the transfected cells, grown on 13-mm coverslips, were placed in a perfused, thermostatted chamber in direct apposition to a low-noise photomultiplier. The photomultiplier has a built-in amplifier-discriminator, the output of which is captured by a Thorn-EMI photon counting board and stored on an IBM-compatible computer for later analysis. Aequorin luminescence data was converted into [Ca2+] values using an algorithm described previously (23.Brini M. Marsault R. Bastianutto C. Alvarez J. Pozzan T. Rizzuto R. J. Biol. Chem. 1995; 270: 9896-9903Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 26.Montero M. Brini M. Marsault R. Alvarez J. Sitia R. Pozzan T. Rizzuto R. EMBO J. 1995; 14: 5467-5475Crossref PubMed Scopus (265) Google Scholar). In all experiments, L max was obtained by integrating a continuous recording of aequorin-mediated light emission in the presence of 0.3% Triton X-100 and 10 mmCa2+. Light output from unstimulated transfected cells loaded with coelenterazine was not significantly higher than background. The light emission from EHA- or MHA-transfected cells loaded in the absence of coelenterazine or from cells transfected with vector alone was not detected. To induce CCE, 100 nm TG or 100 μm histamine were used as indicated in the figure legends. Average cellular [Ca2+]i in PAEC was fluorometrically measured using a Ca2+-sensitive fluorescent dye, fura-2. PAEC were loaded with 2 μmfura-2 for 30 min, washed, and resuspended in nominally Ca2+-free buffer. 5 × 106 cells were used for each measurement. TG was given as indicated. Fluorescence intensity at emission wavelength of 510 nm in response to excitation wavelengths of 340 and 380 nm was determined with the LS50B Spectrophotometer, and [Ca2+]i values were calculated according to the formula of Grynkiewkz et al. (27.Grynkiewkz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar). To assess NO release from intact cells, NOx (NO2− + NO3−) in the culture media was measured with a chemiluminescence NO analyzer (Model 205, Sievers Instruments, Inc., Boulder, CO) as described previously by Wang et al.(10.Wang Y. Shin W.S. Kawaguchi H. Inukai M. Kato M. Sakamoto A. Uehara Y. Miyamoto M. Shimamoto N. Korenaga R. Ando J. Toyo-oka T. J. Biol. Chem. 1996; 271: 5647-5655Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). PAEC or transfected COS-7 cells were incubated in the presence or absence of 100 nm TG in Krebs-HEPES buffer (99 mm NaCl, 4.69 mm KCl, 1.87 mmCaCl2, 1.2 mm MgSO4, 25 mm NaHCO3, 1.2 mmK2HPO4, 11.1 mm glucose, and 20 mm HEPES, pH 7.4) supplemented with 100 μm l-arginine at 37 °C in 95% O2 plus 5% CO2. At the end of incubation, media was collected and 5–10 μl of each sample was used for NO measurement. The amount of NOx was normalized to the protein content determined by Bradford assay. To directly assess TG-mediated NO production, PAEC or transfected COS-7 cells on 25-mm coverslips were cultured in serum-free media for 24 h and loaded with 10 μm DAF-2 DA (Calbiochem), a membrane-permeable fluorescent NO indicator (28.Nakatsubo N. Kojima H. Kikuchi K. Nagoshi H. Hirata Y. Maeda D. Imai Y. Irimura T. Nagano T. FEBS Lett. 1998; 427: 263-266Crossref PubMed Scopus (338) Google Scholar, 29.Kojima H. Nakatsubo N. Kikuchi K. Kawahara S. Kirino Y. Nagoshi H. Hirata Y. Nagano T. Anal. Chem. 1998; 70: 2446-2453Crossref PubMed Scopus (1188) Google Scholar), in Krebs-Ringer phosphate buffer (120 mm NaCl, 4.8 mm KCl, 0.54 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose, and 15.9 mm sodium phosphate, pH 7.2) at 37 °C for 1 h. Cells were washed and placed in 1 ml of Krebs-Ringer phosphate buffer. After 1 min equilibration, TG was added to a final concentration of 100 nm followed by addition of 4 mm Ca2+. Fluorescence was measured with a fluorescence microscope (Olympus IMT-2, Tokyo, Japan) calibrated for excitation at 485 nm and emission at 520 nm. The linearity of the response of DAF-2 to NO was assessed in vitro by generating known concentrations of NO with the NO donor NOC-9 (Calbiochem), measuring fluorescence intensity changes in the presence of 5 mm DAF-2. NOC-9 has a half-life of 3 min, and thus, solutions containing DAF-2 and 3 nm to 1 μm NOC-9 were studied 15 min after mixing in phosphate-buffered saline, representing >95% release of NO from NOC-9. Consistent with the prior publication of Kojima et al. (29.Kojima H. Nakatsubo N. Kikuchi K. Kawahara S. Kirino Y. Nagoshi H. Hirata Y. Nagano T. Anal. Chem. 1998; 70: 2446-2453Crossref PubMed Scopus (1188) Google Scholar) we found that DAF-2 had a linear response in the physiological range of 0–2.4 μm NO (data not shown). Comparisons between groups were made using either paired or unpaired students t test, or ANOVA with Fisher post-hoc test. Data are presented as mean ± S.E., withp < 0.05 accepted as significant. To assess the Ca2+ source for eNOS activation, eNOS-aequorin chimeric constructs were generated by attaching HA epitope-tagged aequorin to the COOH terminus of wild type bovine eNOS (EHA) or myr−-eNOS (MHA) (Fig.1 A), and the chimera were subsequently transfected into COS-7 cells. The recombinant proteins were identified by both eNOS monoclonal antibodies (Fig. 1 B) and HA antibody (data not shown). In contrast to eNOS (M r = 135,000), transfection of COS-7 cells with EHA or MHA resulted in detection of a single product ofM r = 150,000. To determine if the recombinant proteins were properly targeted and retained NOS activity, enzymatic activity assays were performed in subcellular fractions of COS-7 cells transfected with wild type eNOS and myr−-eNOS as well as chimeric EHA and MHA constructs. Equivalent NOS activity was detected in whole cell lysates from cells transfected with all four constructs (Fig. 2 A). Similar to wild type eNOS, NOS activity was 2.5-fold higher in the plasma membrane fraction than that in the cytosol from EHA-transfected cells (Fig.2 B). In contrast, NOS activity was primarily detected in cytosolic and intracellular membrane fractions from either myr−-eNOS or MHA-transfected cells (Fig. 2 B), at a level that was 3–4-fold greater than that in the plasma membrane fraction (Fig. 2 C). Overall, these results indicate that targeting of the chimeric EHA and MHA proteins is similar to that of eNOS and myr−-eNOS, respectively, in transfected COS-7 cells. In addition, NOS enzymatic activity is comparable in EHA- and MHA-transfected cells.Figure 2NOS activity in COS-7 cells transfected with various eNOS constructs. A, NOS activity was assessed by measuring conversion of [3H]l-arginine to [3H]l-citrulline in postnuclear supernatant from cells transfected with eNOS, myr−-eNOS, EHA, or MHA constructs, respectively. 10 μg of protein was used for each assessment. Data are expressed as mean ± S.E. (n= 4). (p > 0.05 versus eNOS.) Enzyme activity was not significantly different among the four samples.B, cytosolic and internal membrane (IM) eNOS activity. Wild type eNOS and EHA constructs had little detectable eNOS activity, whereas myr−-eNOS and MHA constructs had significantly greater (p < 0.001) activity in both compartments. C, ratio of plasma membrane to intracellular (cytosolic plus IM) NOS activity. Wild type eNOS and EHA constructs targeted expression preferentially to the plasma membrane, whereas myr−-eNOS and MHA constructs targeted expression to the intracellular compartments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To localize aequorin activity in transfected cells, the cytosolic fraction was separated from the cell membrane fraction from cells transfected with EHA or MHA constructs. Consistent with the findings for NOS enzymatic activity (Fig. 2), 75% of the EHA luminescence was detected in the plasma membrane fraction, whereas 92% of the MHA activity was detected in the cytosolic fraction. The luminescence activity of EHA protein was not significantly different from that of MHA or ACVI/HA/AEQ chimera (data not shown), indicating that fusing the eNOS cDNA to the NH2 terminus of aequorin did not alter the ability of aequorin as a Ca2+ sensor. In order to measure [Ca2+]i with aequorin, anin vitro calibration curve was generated by measuring luminescence activity in response to differing amounts of EGTA-determined free Ca2+ (Fig.3). The plasma membrane fraction of EHA-transfected cells and the cytosolic fraction of MHA-transfected cells were used for the calibration. The relationship between [Ca2+] and log (L/L max) for EHA was not significantly different from that for MHA (Fig. 3) or from that for ACVI/HA/AEQ, adenylyl cyclase VI-aequorin fusion protein (data not shown). The results indicate that these aequorin constructs measure [Ca2+]i in the physiological range of 10−7 to 10−5m. It has been shown that TG activates CCE subsequent to intracellular Ca2+ pool depletion by inhibiting the microsomal Ca2+-ATPase (31.Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar, 32.Holda J.R. Blatter L.A. FEBS Lett. 1997; 403: 191-196Crossref PubMed Scopus (128) Google Scholar). To determine if the membrane-associated EHA can detect the [Ca2+]ichange resulting from CCE, COS-7 cells were transfected with EHA or MHA constructs and subsequently treated with TG in the absence or presence of extracellular Ca2+ (TableI). The addition of TG resulted in a transient increase in [Ca2+]i in cells transfected with EHA or MHA. No change in luminescence was detected in cells transfected with vector alone (data not shown). Table Idemonstrates that, similar to the previous finding that membrane-bound aequorin primarily sensed Ca2+ influx while the cytosolic aequorin sensed Ca2+ release (16.Nakahashi Y. Nelson E. Fagan K. Gonzales E. Guillou J.-L. Cooper D.M.F. J. Biol. Chem. 1997; 272: 18093-18097Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), MHA tended to detect slightly higher [Ca2+]i resulting from TG-mediated intracellular release than EHA (n = 7,p = 0.13), whereas EHA sensed higher [Ca2+] in response to the addition of 4 mmextracellular Ca2+ than MHA (n = 7,p = 0.04). For EHA, Ca2+ influx resulted in greater [Ca2+]i compared with intracellular release (p < 0.005). Importantly, theL max value was determined in each experiment and the luminescence value was normalized to L max to control for variable luminescence activity resulting from different levels of protein expression. Therefore, the differences detected between EHA and MHA solely reflects [Ca2+]i at the region where the recombinant protein EHA or MHA is distributed, rather than any variation in the level of expression of the two proteins.Table IComparison of intracellular Ca 2+ release and CCE in cells transfected with EHA and MHA constructsTGTG + Ca2+EHA1.29 ± 0.074.31 ± 0.73p < 0.005MHA1.58 ± 0.102.80 ± 0.25p = 0.03p = 0.13p = 0.04[Ca2+]i (μm) in transfected cells was obtained by transforming luminescence activity in response to 100 nm TG stimulation in the presence or absence of 4 mm Ca2+. Data are presented as mean ± S.E. (n = 7). Open table in a new tab [Ca2+]i (μm) in transfected cells was obtained by transforming luminescence activity in response to 100 nm TG stimulation in the presence or absence of 4 mm Ca2+. Data are presented as mean ± S.E. (n = 7). In order to determine whether a physiological agonist could give rise to the differences in [Ca2+]i reported by the two aequorin chimeras in response to release versus CCE, histamine, an agonist stimulating intracellular Ca2+release via the generation of inositol 1,4,5-triphosphate (33.Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar) was explored. To evoke the release of stored Ca2+, cells were stimulated with histamine in the absence of external Ca2+; this was followed by the addition of external Ca2+ (Fig.4). The increase of [Ca2+]i was smaller in the absence than presence of [Ca2+]ex in EHA-transfected cells,i.e. 2 versus 5.5 μm, whereas the increase of [Ca2+]i was the same in the absence or presence of extracellular Ca2+ in MHA-transfected cells, about 2.8 μm. Thus, membrane-bound EHA was more sensitive to CCE while cytosol" @default.
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