Somatostatin Receptor Subtype-Dependent Regulation of Nitric Oxide Release: Involvement of Different Intracellular Pathways

Sara Arena1, Alessandra Pattarozzi1, Alessandro Corsaro, Gennaro Schettini and Tullio Florio

Section of Pharmacology, Department of Oncology, Biology and Genetics, University of Genova, and Pharmacology and Neuroscience, National Institute for Cancer Research (IST), 16132 Genova, Italy

Address all correspondence and requests for reprints to: Professor Tullio Florio, Sezione Farmacologia, Dipartimento Oncologia, Biologia e Genetica, Università di Genova, Largo Rosanna Benzi 10, 16132 Genova, Italy. E-mail: florio{at}cba.unige.it.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We reported previously that, in addition to direct effects, somatostatin (SST) affects tumor growth inhibiting the tumoral neoangiogenesis, via an interference with NO synthesis. Here, we analyzed the effects of SST on nitric oxide (NO) production induced by different agonists [basic fibroblast growth factor (bFGF), insulin, cholecystokinin (CCK)] and the intracellular signaling involved, using Chinese hamster ovary-k1 cells stably transfected with individual SSTR1–SSTR4. bFGF and insulin induced endothelial nitric oxide synthase activity via the generation of ceramide or the Akt-dependent phosphorylation of endothelial nitric oxide synthase, respectively. CCK regulates neuronal nitric oxide synthase activity in a Ca++- dependent manner. SST inhibited NO production stimulated by bFGF through SST receptor 1 (SSTR1), SSTR2, and SSTR3 and by CCK through SSTR2 and SSTR3. In all the cell lines, SST treatment did not modify NO synthesis induced by insulin. SSTR4 activation was not effective on any of the stimuli tested. The effects on bFGF-induced NO production were downstream from receptor phosphorylation and ceramide synthesis. SSTR2 and -3 on CCK activity were related to the inhibition of intracellular Ca++ mobilization, whereas the lack of effects on insulin was paralleled by the absence of SST activity on Akt phosphorylation. These data, identifying for the first time a selective receptor subtype-inhibitory role of SST on NO generation, may open new perspectives in the use of SST agonists to control tumoral angiogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NITRIC OXIDE (NO) is a unique intra/intercellular messenger involved in the regulation of many physiological functions such as central and peripheral neurotransmission, smooth muscle contractility, platelet reactivity, and angiogenesis, among others (1). An inappropriate production of NO has been linked to a number of pathologies; therefore, compounds that modulate its release may display a great therapeutic value (2).

NO is generated by a family of enzymes known as NO synthase (NOS), named after the tissues in which they were originally described. However, endothelial and neuronal NOS (eNOS, nNOS) are constitutively expressed not only in endothelia and neurons but also in many other cell types (3). They release NO in response to receptor stimulation after a Ca++/calmodulin-dependent pathway (1). More recently, different kinds of stimuli have been proved to activate eNOS in a Ca++-calmodulin-independent way. We have recently reported that basic FGF (bFGF), similar to what was reported for bradykinin (4), is able to activate eNOS in Chinese hamster ovary (CHO)-k1 cells, via the sphingomyelinase-dependent generation of ceramide, causing the translocation of eNOS from the plasma membrane, where it is bound to caveolin-1, to the cytosol in its active form (5).

Insulin has also been reported to induce NO synthesis in a Ca++-independent manner, through the Akt-dependent phosphorylation and activation of eNOS (6). Thus, it is now well recognized that the constitutive NOS isoforms may be activated in both Ca++/calmodulin-dependent and -independent manner. The third isoform of the enzyme, inducible NOS, is regulated transcriptionally, being induced upon stimulation of cytokines and other proinflammatory agents (3).

Among the different roles attributed to NO in physiology and pathology, its involvement in carcinogenesis and angiogenesis has been recently achieving great importance (7, 8, 9). NO has been reported to induce vascular endothelial growth factor expression in carcinoma cells (10) and neovascularization in tumors (11). Moreover, angiogenic factors, including both bFGF and vascular endothelial growth factor, are powerful NOS activators (5, 8). Thus, the identification of compounds with antiangiogenic properties is acquiring a pivotal relevance for the inhibition of tumor growth, and substances able to block NO production may be active in this respect.

Somatostatin (SST) is an endogenous peptide that exerts a direct antiproliferative activity in different neuroendocrine (12, 13) and epithelial tumors (14). More recently, we and others demonstrated its capability to act as a pure antiangiogenic factor in in vivo and in vitro experiments (15, 16, 17). Moreover, we demonstrated, for the first time, the capability of SST to inhibit eNOS activity in endothelial cells showing that this effect caused the inhibition of neoangiogenesis and tumor growth (17).

The aim of this work was to characterize the SST-inhibitory effects on NO production and to identify the SST receptor (SSTR) subtypes responsible for such effects and the intracellular pathways involved. To achieve this goal, we used CHO-k1 cells transfected with individual SSTR subtypes 1, 2, 3, and 4. The choice of this experimental model was taken on the basis of the following considerations: 1) CHO-k1 cells express multiple NOS isoforms (namely eNOS and nNOS)(18, 19), thus allowing the analysis of the effects of different NOS isoenzymes; 2) NO generation can be induced through different intracellular pathways using bFGF, insulin, or cholecystokinin (CCK) as agonists (18, 19), thus allowing the evaluation of specific interaction among SSTRs and multiple second messenger systems; and 3) the expression of significant levels of the individual SSTR subtypes allows the identification of the selective role and signal transduction of each of them.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of bFGF, Insulin, and CCK on eNOS and nNOS Activity in CHO-k1 Cells and Intracellular Mechanisms Involved
We analyzed NO released from CHO-k1 cells after bFGF, insulin, and CCK treatment, measuring NOS activity as conversion of [3H]-L-arginine in [3H]-L-citrulline. Both bFGF and insulin caused a significant increase in NOS activity starting after 1 h of treatment and becoming maximal after 2 h, with bFGF more efficacious than insulin (Table 1Go). Interestingly, although slowly declining, the enzymatic activity lasted up to 18 h of treatment (Table 1Go). CCK treatment resulted in a highly significant NOS activation (about +200% of basal activity) as early as after 30 min, reached a peak after 1 h, and then slowly declined although remaining still effective after 18 h of treatment (Table 1Go). We then performed the following experiments using the Griess reaction that evaluates NOS activity by nitrite/nitrate accumulation in the cell culture medium. Because this assay requires longer incubation times to be sensitive, we performed the experiments after 18 h of treatment, to maximize the NO production. Figure 1Go shows the dose-response curves of bFGF, insulin, and CCK in CHO-k1 cells. bFGF caused NO accumulation in the cell medium, beginning at the concentration of 5 ng/ml and reaching a maximal effect at 100 ng/ml (max. {approx}+200%); insulin also stimulated NO production in a dose-dependent manner beginning at 10 nM up to 500 nM (max. {approx}+100%), whereas CCK led to NO synthesis in a range of concentration between 20 µM and 100 µM (max. {approx}+400%).


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Table 1. Time-Course of the Effects of bFGF, CCK, and Insulin on NOS Activity in CHO-k1 Cells

 


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Fig. 1. Dose-Response of bFGF (A), Insulin (INS) (B), and CCK (C) on NO Production

Cells were treated for 18 h with different concentrations of the compounds. Data are expressed as nitrate/nitrite accumulation, measured as OD (absorbance at 570 nm). **, P < 0.01 vs. respective basal values.

 
It was reported previously that CHO-k1 cells express both the endothelial and the neuronal isoforms of NOS, whereas the inducible one was detected neither in basal nor in stimulated conditions (18, 19). We confirmed the presence of the constitutive isoforms of NOS by both RT-PCR and Western blot experiments (Fig. 2AGo). To better define which of the constitutive isoforms of NOS was responsible for the NO production subsequent to the stimulation with the different agonists, we looked for the enzymatic activity after immunoprecipitating the cells, pre-treated with bFGF, CCK, and insulin, with {alpha}-eNOS and {alpha}-nNOS antibodies. Lysates from control cells, and from cells treated with the different compounds, were immunoprecipitated with the specific {alpha}-eNOS and {alpha}-nNOS antibodies, and an enzymatic assay for NOS activity was performed on the samples, measuring the conversion of [3H]-L-arginine in [3H]-L-citrulline.



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Fig. 2. Selectivity of the Effects of bFGF, CCK, and Insulin (INS) on eNOS and nNOS Isoforms

A, RT-PCR (upper panel) and Western blot (lower panel) detection of eNOS and nNOS mRNA and protein expression in CHOk1 cells (lanes 2 and 4). Human umbilical vein endothelial cells (lane 1) and rat brain extracts (lane 3) were used as positive controls for eNOS and nNOS, respectively. B, Specific agonist activation of the NOS isoforms in CHO-k1 cells. NOS activity was measured after immunoprecipitation with eNOS and nNOS antibodies. Cells were serum starved for 24 h and then treated for 18 h with bFGF (50 ng/ml), CCK (20 µM), and INS (200 nM). Then, equal amounts of protein (100 µg) from each cell lysate were immunoprecipitated with anti-eNOS and anti-nNOS antibodies. NOS activity was measured on the immunoprecipitate evaluating the conversion of [3H]-L-arginine in [3H]-L-citrulline. Data are expressed as percentage of the respective basal activity. **, P < 0.01 vs. respective basal values.

 
bFGF caused a strong increase in eNOS activity (Fig. 2BGo), with an effect that was already statistically significant after 10 min of treatment and lasted up to 24 h (data not shown), whereas no changes were observed in nNOS activity (Fig. 2BGo), suggesting the exclusive involvement of the endothelial isoform in the bFGF-mediated NO production, in CHO-K1 cells. Conversely, CCK treatment led to a significant activation of nNOS, whereas it did not influence eNOS activity, thus showing that CCK treatment induces NO production through nNOS activation (Fig. 2BGo). The enzymatic assay performed after treatment with insulin resulted in a significant increase in eNOS activity without affecting that of the nNOS isoform (Fig. 2BGo). Thus, we show that both NOS isoforms detectable in CHO-K1 cells are involved in the synthesis of NO, with eNOS being preferentially activated by tyrosine kinase receptors (bFGF, insulin), whereas nNOS is mainly regulated by the G protein-coupled receptor CCK-A.

To better dissect the intracellular mechanisms involved in the pathways leading to NO synthesis after activation of these three receptor systems, we induced NO production by treating the cells with bFGF, insulin, and CCK together with different compounds that selectively inhibit the different possible pathways involved. In particular, according to previous studies (5, 6, 18), we analyzed the role, in the NO production, of the ceramide synthesis via sphingomyelinase activation, of the changes in intracellular Ca++ concentration ([Ca++]i) and of the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade that leads to the phosphorylation and activation of eNOS. To block the ceramide-dependent activation of eNOS, we used desipramine (10 µM), an inhibitor of acidic sphingomyelinase (20); to block the Ca++-calmodulin activation of NOS, we used the cell-permeable Ca++ chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetra(acetoxymethyl)ester BAPTA-AM (20 µM); and to inhibit the eNOS activation by Akt, we used Ly 294002 (10 µM), a selective inhibitor of the PI3K-dependent Akt phosphorylation.

The results, represented in Fig. 3Go, show that each inhibitor used was able to selectively interfere with a specific receptor stimulus. Desipramine completely inhibited NO synthesis when coincubated with bFGF whereas it was not able to affect the NO synthesis stimulated by CCK and insulin. The Ca++-chelator BAPTA-AM blocked the production of NO only when it was stimulated by CCK, confirming that this peptide acts through a Ca++-dependent activation of NOS without interfering with the activation of the enzyme by bFGF and insulin. Finally the insulin-dependent NOS activation was abolished by blocking the PI3K-Akt cascade. Conversely, the cotreatment with Ly 294002 did not affect NO production induced by bFGF and CCK. No significant changes in basal NO production were observed using each of the above mentioned inhibitors in the absence of agonists (Fig. 3Go).



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Fig. 3. Specificity of the Intracellular Pathways Leading to the NO Production Dependent on bFGF, CCK, and Insulin (INS) in CHO-k1 Cells

The intracellular pathways mediating NO production by bFGF, CCK, and insulin (INS), were dissected using pharmacological inhibitors of the different intracellular messengers involved. Cells, serum starved for 24 h, were treated for 18 h with bFGF (50 ng/ml), CCK (20 µM), INS (200 nM) in the presence or absence of the acidic sphingomyelinase inhibitor desipramine (DES, 10 µM), the cell-permeable Ca++ chelator BAPTA-AM (20 µM), and the selective inhibitor of the PI3K-dependent Akt phosphorylation Ly 294002 (LY, 10 µM). Data are expressed as nitrate/nitrite accumulation, measured as OD (absorbance at 570 nm) and expressed as percentage of control value. Control value was OD570 0.191 corresponding to 7.8 ± 0.3 nmol/300 µl of nitrites. **, P < 0.01 vs. control values; °°, P < 0.01 vs. stimulated values.

 
Thus, in CHO-k1 cells, the stimulation of three different receptors is able to activate the same enzyme, although in two different isoforms, by completely distinct intracellular pathways.

Effects of SSTR1–4 Activation on NO Production
Because NO is recognized to play a central role in the angiogenic process, we analyzed the possible interference of SST with agonist-induced NO synthesis, as this peptide has been shown to inhibit tumoral neovascularization (17).

To investigate whether SST was able to interfere with NO synthesis in CHO-k1 cells, we generated stable cell lines transfecting the cDNA of the rat isoforms of SSTR1, -2, -3, and -4 in CHO-k1 cells (CHO-SSTR1, CHO-SSTR2, CHO-SSTR3, and CHO-SSTR4, respectively). These four cell lines express comparable levels of SSTR (Table 2Go), allowing us to study the effects of the individual SSTR subtypes. In agreement with previous reports (21, 22), all four SSTR subtypes studied were able to induce the phosphorylation/activation of ERK1/2, showing that, in addition to the correct expression, the transfected receptors were all functioning (Fig. 4Go).


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Table 2. Characterization of Kd and Bmax Values of the SSTR-Expressing Cell Lines

 


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Fig. 4. SST Induces ERK1/2 Phosphorylation in CHO-SSTR1–4

Representative Western blot showing ERK1/2 phosphorylation-activation subsequent to SST treatment (1 µM, 10 min) in CHO-SSTR1, CHO-SSTR2, CHO-SSTR3, and CHO-SSTR4 (upper panel). Equal protein loading was demonstrated probing the membrane with an antibody directed against the total ERK1/2 (lower panel). In the graph is reported the ratio of the densitometric analysis of phospho-ERK1/2 and the total ERK1/2 gels, expressed as folds of increase of SST treatment over the values of untreated control cells, as average of three independent experiments.

 
Basal NO production in CHO-K1 cells was quite reproducibly around 8 nmole/300 µl during 18 h of experiment, and the transfection of individual SSTR did not significantly modify this value. Moreover, in all the cell lines, SST treatment did not affect basal NO synthesis (data not shown); thus we checked the effects of the peptide in NOS-stimulated conditions.

Figure 5AGo shows the dose-response curve of SST on the bFGF-mediated nitrite/nitrate accumulation in CHO-SSTR1, -2, -3, and -4. SST treatment caused a dose-dependent decrease of bFGF-dependent NO accumulation in CHO-SSTR1, -2, and -3, whereas it was completely ineffective in CHO-SSTR4. The activation of SSTR1, -2 and -3 by SST caused a decrease in the NO production that was similar in magnitude and potency among the different cell lines (Fig. 5AGo). SST effect was statistically significant starting at the concentration of 300 nM and maximal at the concentration of 3–10 µM. SST inhibition of NO production was mimicked, only in the cell lines expressing the respective receptor, by the treatment with the SSTR1 agonist BIM23745 by the SSTR2/5 agonist lanreotide, and by the SSTR3 agonist L796778 (data not shown). Interestingly, SST effects were completely reverted by pretreatment with pertussis toxin (PTX) (180 ng/ml, 18 h)(Fig. 5BGo), indicating that, similar to almost all the other effects mediated by SSTR1, -2 and -3, the regulation of NO production was also mediated by a G protein of the Gi/o family.



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Fig. 5. Effects of SST on bFGF-Induced NO Synthesis

A, Dose response of SST on bFGF-stimulated NO synthesis in CHO-SSTR1–4. Cells were treated for 18 h with bFGF (50 ng/ml) and with increasing concentration of SST (added 10 min before bFGF), and NO accumulation was evaluated with the Griess reaction. Data are expressed as a percentage of the bFGF-stimulated nitrate/nitrite accumulation measured as OD (absorbance at 570 nm). Basal values (nmol/300 µl) were: 6.9 ± 0.2 for CHO-SSTR1, 7.2 ± 0.25 for CHO-SSTR2, 7.8 ± 0.3 for CHO-SSTR3, and 7.9 ± 0.5 for SSTR4. bFGF increased basal NO production by 120% in CHO-SSTR1 cells, 95% in CHO-SSTR2 cells, 104% in CHO-SSTR3 cells, and by 89% in CHO-SSTR4 cells. °, P < 0.05; and °°, P < 0.01 vs. bFGF stimulated values. B, Role of G protein in the SST inhibition of bFGF-dependent NOS activity in CHO-SSTR1, CHO-SSTR2, and CHO-SSTR-3. Control or PTX-pretreated (180 ng/ml, 18 h) cells were treated for 18 h with bFGF (50 ng/ml) in the presence or absence of SST (10 µM, added 10 min before bFGF). NO accumulation was then evaluated with the Griess reaction. Data are expressed as nitrate/nitrite accumulation measured as OD (absorbance at 570 nm). **, P < 0.01 vs. respective control values; °°, P < 0.01 vs. bFGF-stimulated values.

 
Next we analyzed the SST effects on NO production stimulated by CCK or insulin. SST significantly reduced NO synthesis induced by CCK in CHO-SSTR2 and in CHO-SSTR3, but it had no effect on CHO-SSTR1 and -4 (Fig. 6AGo). This effect was completely abolished by PTX pretreatment, confirming, also for the inhibitory effects of SST on CCK-dependent NO production, a role for a G protein of the Gi/o family (Fig. 6BGo). As for the insulin-dependent increase in NO production, SST was not able to affect it in any of the transfected cell lines, indicating that this peptide is unable to interfere with the intracellular pathway activated by insulin receptor activation (data not shown).



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Fig. 6. Effects of SST on NO Production Stimulated by CCK in CHO-SSTR1–4

A, CHO-SSTR1–4 cells were pretreated with SST (10 µM for 10 min) or PBS and then treated for 18 h with CCK (20 µM). NO production was then evaluated with the Griess reaction. Data are expressed as a percentage of CCK-stimulated nitrate/nitrite accumulation, measured as OD (absorbance at 570 nm). Basal values (nmol/300 µl) were: 8.1 ± 0.5 for CHO-SSTR1, 8.2 ± 0.6 for CHO-SSTR2, 7.8 ± 0.6 for CHO-SSTR3, and 8.0 ± 0.8 for SSTR4. CCK increased basal NO production by 79% in CHO-SSTR1 cells, 86% in CHO-SSTR2 cells, 101% in CHO-SSTR3 cells, and by 76% in CHO-SSTR4 cells. °°, P < 0.01 vs. respective stimulated values. B, Role of G protein in the SST inhibition of CCK-dependent NOS activity in CHO-SSTR2–3. CHO-SSTR2 and CHO-SSTR3 cells were pretreated with PTX (180 ng/ml, 18 h) and then treated for 18 h with CCK (20 µM) in the presence or absence of SST (10 µM, added 10 min before CCK). NO accumulation was then evaluated with the Griess reaction. Data are expressed as nitrate/nitrite accumulation measured as OD (absorbance at 570 nm). **, P < 0.01 vs. respective control values; °°, P < 0.01 vs. CCK-stimulated values.

 
Intracellular Pathways Involved in the SST Regulation of NO Production
To delve deeper into the different intracellular signaling activated by the individual SSTRs responsible for the inhibitory effects of SST on NO production, we tested the effects of SST on ceramide (the second messenger involved in bFGF effects)-induced NO secretion, on the regulation of [Ca++]i induced by CCK, responsible for the CCK-dependent NOS activation, and on the activation of Akt, which mediates the insulin effects.

First we tried to determine whether SST inhibition on bFGF-induced NO could occur before or after the synthesis of ceramide. For this purpose we treated the cells with a cell-permeable C8:0 ceramide in the absence or presence of different concentrations of SST and than we looked for NO production. In CHO-SSTR1, -2, and -3, but not in CHO-SSTR4, SST caused a dose dependent-reduction of NO generation (Fig. 7Go), thus indicating that SST probably acts downstream from the ceramide synthesis. Moreover, the lack of effects observed after SSTR4 activation confirms the results observed in the similar experiments performed using bFGF. Moreover, we analyzed, in CHO-SSTR2 cells, the effects of SST treatment on the intracellular localization of eNOS. Indeed, the inactive form of this enzyme is bound to the membrane in caveolae but, upon activation, it translocates to the cytosol (23). We previously demonstrated that bFGF treatment of CHO-k1 cells causes activation of eNOS via a ceramide-dependent dissociation from caveolin-1 (5). Also in CHO-SSTR1, -2, and -3 cells, bFGF treatment significantly reduced the amount of eNOS bound to caveolin-1, evaluated by Western blot assay after immunoprecipitation with an antibody directed against caveolin-1, as previously reported (24) (Fig. 8Go). Moreover, pretreatment with SST caused a significant reduction of the bFGF effect (Fig. 8Go).



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Fig. 7. Dose Response of SST on Ceramide-Stimulated NO Synthesis in CHO-SSTR1–4

Cells were treated for 18 h with C8:0 ceramide (Cer) (20 µM) and with increasing concentration of SST (added 10 min before ceramide). NO production was measured by the Griess reaction. Data are expressed as a percentage of the ceramide nitrate/nitrite accumulation measured as OD (absorbance at 570 nm). Basal values (nmol/300 µl) were: 7.5 ± 0.4 for CHO-SSTR1, 8.3 ± 0.7 for CHO-SSTR2, 7.9 ± 0.6 for CHO-SSTR3, and 8.2 ± 0.6 for SSTR4. Cer increased basal NO production by 66% in CHO-SSTR1 cells, 59% in CHO-SSTR2 cells, 52% in CHO-SSTR3 cells, and by 48% in CHO-SSTR4 cells. °, P < 0.05; and °°, P < 0.01 vs. respective ceramide-stimulated values.

 


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Fig. 8. SST Treatment Prevents the bFGF-Dependent eNOS Delocalization from Caveolin 1

Upper panel, CHO-SSTR2 cells were plated in 10-mm dishes and left untreated (C, lane 1) or treated for 1 h with bFGF (50 ng/ml) (FGF, lane 2) and bFGF + SST (10 µM) (F+SST, lane 3). Equal amounts of proteins from total cell lysate (250 µg) were immunoprecipitated with anticaveolin-1 (cav-1) antibody. Western blot was performed using either anti-eNOS (upper lane) or anticaveolin-1 (lower lane) antibodies, the latter to demonstrate that equal amounts of caveolin-1 were immunoprecipitated. Lower panel, Quantitation of eNOS dissociation from caveolin-1 in CHO-SSTR2, as well as in CHO-SSTR1 and -3 (Western blot not shown) performed by densitometric analysis of three independent experiments from each cell line.

 
To identify the interference of SSTR with the intracellular pathway activated by CCK-A receptor, we evaluated the changes in [Ca++]i in microfluorimetric experiments at the single-cell level, using CHO-SSTR1, -2, and -3-transfected cells. As shown in Fig. 9Go, the activation of the different receptor subtypes had a completely opposite response to SST (10 µM). In fact, whereas the peptide totally blocked Ca++ mobilization subsequent to CCK-A stimulation in CHO-SSTR2 and -3, it had no effect on CHO-SSTR1, thus confirming the results obtained with the nitrite/nitrate assay. Therefore, we propose that SST interferes with NO production induced by CCK-A activation, preventing the intracellular Ca++ mobilization necessary for NOS activation, via the SSTR2 and -3-regulated intracellular signaling.



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Fig. 9. Effects of SST on CCK-Induced [Ca++]i Mobilization in CHO-SSTR1, CHO-SSTR2, and CHO-SSTR3

[Ca++]i was measured using the fluorescent probe Fura2, in microfluorimetry. Cells were treated with CCK (20 µM) after a 1-min pretreatment with SST (10 µM) or equal volume of PBS, as indicated by the arrows. At least 12 cells per field were analyzed, and the data are expressed as mean values.

 
Concerning the effects of SST on the production of NO induced by insulin, we checked whether SSTR activation could modulate the insulin-dependent Akt phosphorylation/activation, because in our experimental model (see Fig. 3Go), as well as in other cell types (6), this is the main intracellular mechanism involved in NOS activation by this hormone. The Western blot reported in Fig. 10Go shows that no significant differences in the insulin-dependent Akt phosphorylation occurred between cells treated or untreated with SST. In fact, the peptide was not able to block or to prevent Akt phosphorylation subsequent to insulin treatment in any of the SSTR subtype-expressing cell line (Fig. 10Go). This observation is in agreement with the previously described experiments, in which SST was unable to decrease insulin-dependent NO production.



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Fig. 10. Lack of Effects of SST on Insulin-Induced Akt Phosphorylation in CHO-SSTR1–4

Left panels, SST effects on Akt activation were evaluated as Ser (473)-phosphorylation in Western blot, using a phospho-specific antibody. CHO-SSTR1–4 were pretreated with SST (10 µM, 10 min) and then treated with insulin (INS) (200 nM) for 5 min. Right panels, Equal protein loading was demonstrated probing the membrane with an antibody directed against the total Akt protein.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NO is now recognized as one of the major regulators of the angiogenic process in both physiological and pathological conditions. In particular, the antiangiogenic therapy has been proposed as an important new approach for the pharmacological treatment of tumors (25, 26). Thus, the pharmacological regulation of NO release may represent a possible significant target for antiangiogenic therapy (2, 7). In the past years, in addition to a direct antiproliferative activity (27, 28), SST was proposed to affect tumor growth through indirect mechanisms including the inhibition of tumoral neoangiogenesis (15, 16, 17). In particular, in in vitro and in vivo experiments, we demonstrated that this activity was dependent on the regulation of both endothelial cell proliferation and invasion, and monocyte activation and migration (16). Importantly, we recently reported that the antiangiogenic activity of SST was dependent on the regulation of both eNOS and MAPK activities, with the inhibition of the former being a prerequisite for the in vivo effects of SST (17). However, the intracellular pathways mediating the SST regulation of NOS activity and the SSTR subtype specificity of such an effect have not yet been determined. Thus, the aim of this study was to identify the SSTR subtypes and the intracellular pathways involved in the SST regulation of NO production.

We took advantage of CHO-k1 cells in which NO synthesis can be induced by multiple agonists through the modulation of independent intracellular mechanisms, acting on different NOS isoforms. In fact, we demonstrated that, in agreement with a previous report (18), the classical Ca++-dependent NO production was mediated by the activation of nNOS through the G protein-coupled receptor CCK-A, whereas the bFGF and insulin tyrosine kinase receptors preferentially regulate eNOS in a Ca++-independent way, involving the activation of sphingomyelinase and PI3K/Akt, respectively. The relevance of these results reside in the demonstration of how, in the same cellular background, independent pathways are able to connect different agonist/receptor systems with a common physiological function, such as NO release. By expressing individual SSTR1, -2, -3, and -4 in these cells, we were able to test the effects of the single SSTR activation on the independent mechanisms regulating NO production. Since the molecular cloning of the members of the SSTR family, most of the studies focused on the identification of specific biological functions and biochemical pathways attributed to each of the SSTR subtypes in cells expressing either native or heterologous SSTR. However, in both experimental systems, a large overlap of functions has been discovered. This is true not only for predicted SSTR activities (i.e. the inhibition of cAMP accumulation) (28) but also for more novel and unexpected functions, such as the activation of ERK1/2 in CHO-k1 cells (Refs. 21 and 22 and present paper) or the activation of the SHP2 tyrosine phosphatase (29, 30). A partially different scenario occurred in our cell system because we found only a partial overlap in the SSTR1–4 effects on SST regulation of NO production. First of all, a completely different response was observed activating SSTR4. Indeed, although the receptors were correctly expressed and active after SST treatment (as determined by ERK1/2 activation), we did not detect any modulation of NO production induced by both Ca++-dependent and independent pathways.

Conversely, the activation of SSTR1, -2, and -3 resulted in a significant inhibition of NO production. Interestingly, significant differences were detected after activation of the different SSTR subtypes, according to the intracellular pathway used to induce NO release. In particular, it is noteworthy that none of the receptors was able to affect NO release induced by insulin treatment, probably because the activation of SSTR1–4 was not able to modify the Akt activation induced by the growth factor. Conversely, a mirror-like effect was observed on the bFGF regulation of eNOS. In these experimental conditions, SSTR1, -2, and -3 significantly reduced the NO production induced by the bFGF-dependent ceramide production.

Although we were not able to identify the exact intracellular mechanism involved in such an effect, the SST interference in the NOS activation was downstream from the ceramide synthesis, because a similar inhibition pattern was observed when NO release was induced by the administration of exogenous, cell-permeable, short-chain ceramide. We demonstrated previously that the ceramide produced after bFGF treatment in CHO-k1 cells caused the activation of eNOS through a relocation of the enzyme from the cell membrane to the cytosol (5). We observe that SST treatment of CHO-SSTR1–3 cells prevents this effect, indicating that the intracellular signaling activated by SST may interfere with the ceramide-dependent NOS activation pathway. Moreover, as reported for most of the intracellular signaling activated by SST (27), also the modulation of eNOS activity by SSTR1–3 was mediated by a G{alpha}i/o PTX-sensitive GTP-binding protein. Although we do not have definitive data, we can speculate that the reactive oxygen species generation caused by ceramide treatment (31) may favor the dissociation of inactive eNOS from caveolin 1, allowing its cytosolic translocation and activation (32) and that somatostatin may act, inhibiting the release of oxygen radicals (33).

Finally, nNOS activity induced by CCK treatment was completely blocked only after SSTR2 and -3 activation whereas the stimulation of SSTR1 was ineffective. Also, in these experiments the SST regulation of NO production was PTX sensitive and correlated with the activating second messenger pathway. Indeed, similar to the observed effects on the CCK-dependent NO regulation, in CHO-SSTR-2 and -3, but not in CHO-SSTR1, SST significantly blocked the intracellular Ca++ rise caused by CCK. It was reported previously reported that in endocrine cells SST may interfere with phospholipase C (PLC) activity to block the intracellular Ca++ increase (34, 35). Although, thus far, contrasting data have been reported about this possibility (36), we cannot exclude that such inhibition of PLC activity may occur also in our experimental model. Further studies will be required to better address this issue. The identification of the intracellular pathways mediating SSTR3 inhibition of NO production is particularly interesting because we recently demonstrated that the activation of this receptor subtype is necessary for the experimental tumoral angiogenesis blockade in vivo and that this activity requires NOS inhibition (17). Here we report that SSTR3 inhibits NO production through both Ca++-dependent and -independent mechanisms, the latter involving the reversal of NOS activation via the growth factor-induced ceramide production.

In summary, as depicted in the cartoon in the Fig. 11Go, we demonstrate that SST is able to affect NO production but that its activity depends on the signaling context that leads to different intracellular pathways and on the SSTR subtype activated. The identification of selective intracellular pathways and SSTR subtypes able to affect NOS activity will represent an important basis on which to evaluate the role of novel and more specific SST analogs in native SSTR- expressing cell types (i.e. endothelial cells) supporting their use as antiangiogenic compounds.



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Fig. 11. Diagrammatic Representation of the Subtype-Specific Effects of SSTRs on NO Production

In the cartoon are depicted the intracellular pathways involved in the bFGF-, CCK-, and insulin (INS)-induced NOS activation in CHO-k1 cells and the interference of SST via the different SSTR subtypes. Insulin modulates NOS activity via the PI3K/Akt-dependent phosphorylation of eNOS; CCK induces a PLC-mediated release Ca++ from the intracellular stores responsible for the classical Ca++-dependent activation of nNOS; bFGF regulates eNOS activity via the activation of sphingomyelinase (SM) and the generation of ceramide. SST via the SSTR subtypes 1, 2, and 3 inhibits the bFGF effects acting downstream from the ceramide production and via SSTR2 and -3 antagonizes the Ca++-dependent NO production induced by CCK. Insulin activation of eNOS is not modified after SST treatment. SSTR4 is the only SSTR subtype tested that was ineffective on all the intracellular pathways leading to NO production.

 
Our data are in contrast to a study by Lopez et al. (37) in which shown an activation of nNOS by SST, via SSTR2, in a different strain of CHO cells (CHO-DG44) was shown. Although we do not have a definitive answer for such a discrepancy, it is very likely that differences of strains (i.e. CHO-k1 vs. CHO-DG44) may play a relevant role in the different results observed. In fact, in that study, the activation of nNOS was dependent on SHP1 activation by SST, and this protein tyrosine phosphatase is not expressed in our cells (21). Similarly, previous studies from the same group showed that in CHO-k1 cells, the same strain of CHO cells that we used in our study, SST caused an inhibition of cGMP production induced by CCK, probably involving an inhibitory effect on NO release (38). A similar discrepancy was reported previously in the regulation of adenylyl cyclase activity: the activation of SSTR1 and -2 resulted in an inhibitory effect in CHO-k1 cells (39), but it was ineffective in CHO-DG44 (40).

In conclusion, we have identified the molecular mechanisms involved in the SST regulation of NOS activity, showing that the activation of SSTR1, -2, and -3, but not SSTR4, is able to affect NO production after heterologous expression in CHO-k1 cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
SST, CCK, bFGF, LY-83583, BAPTA-AM, Fura-2 penta-acetoxymethyl ester were purchased from Calbiochem (Lucerne, Switzerland), vanadate from ICN Biochemicals, Inc. (Cleveland, OH), insulin and desipramine by Sigma (Milano, Italy), C8:0 ceramide from Alexis, [3H]-L-arginine and [131I]-Tyr-SST from Amersham (Milano, Italy).

Antibodies
Anti-eNOS, nNOS, and anticaveolin-1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-ERK1/2 and phospho-ERK1/2, anti-Akt and phospho(Ser 473)-Akt were from New England Biolabs, Inc. (Beverly, MA).

Cell Cultures
CHO-k1 cells were cultured under sterile conditions in F-12 Ham’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) (Invitrogen).

Cloning of Rat (r) SSTR3 and r-SSTR4
r-SSTR3 and r-SSTR4 cDNA were cloned from total rat brain cDNA using a high-fidelity PCR approach. We used the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, IN) containing a mixture of Taq and Pwo DNA polymerases, which ensures a high-fidelity transcription (41).

The sequences of the primers were as follows:

ATA/GAA/TTC/ATG/GCC/GCT/GTT/ACC/TAT/CCT (forward) and ATA/CTC/GAG/TTA/CAG/ATG/GCT/CAG/CGT/GCT/GGC (reverse) corresponding to amino acids 1–7 and 423–428 of r-SSTR3 (GenBank accession no. X63574), and ATA/GAA/TTC/ATG/AAC/ACG/CCT/GCA/ACT/CTG (forward) and ATA/CTC/GAG/TCA/GAA/AGT/AGT/GGT/CTT/GGT/GAA (reverse) corresponding to amino acids 1–7 and 379–384 of r-SSTR4 (GenBank accession no. U04738). A stop codon (underlined) and EcoRI or XhoI restriction sites (double underlined) were introduced in the primers. The PCR protocol used was the following: 3 min at 95 C followed by 20 cycles of 15 sec at 94 C, 30 sec at 62 C, 45 sec at 70 C with 5 sec extension each cycle for the last step and a final step at 72 C for 7 min. The amplified product underwent agarose gel electrophoresis analysis and then subcloned in the plasmid pCDNA3 (Pharmacia Biotech, Piscataway, NJ) using the EcoRI and XhoI restriction sites introduced in the primers used for the PCR.

The subcloned cDNAs were then sequenced (Primm, Milano, Italy), confirming the identity between the amplified cDNA and the published r-SSTR3 and r-SSTR4 sequences.

Generation of Stable Cell Lines
The generation of CHO-k1 cells stably expressing r-SSTR1 and r-SSTR2A (CHO-SSTR1 and CHO-SSTR2, respectively) has been previously described and characterized (39, 42). CHO-SSTR3 and CHO-SSTR4 were generated by stable transfection of CHO-k1 cells with a plasmid containing the open-reading frames of r-SSTR3 and r-SSTR4 in the PCDNA3 vector using the Fugene reagent (Roche), according to the manufacturer’s instruction. Transfectants were isolated by growing the cells in a culture medium supplemented with G418 (Sigma) (500 µg/ml). Single colonies were then isolated and expanded, and receptor expression was demonstrated in binding experiments using [131I]-Tyr-SST (Amersham) (39). Briefly, saturation binding assay were performed using [131I]-Tyr-SST (5–500 pM) in the presence or absence of 100 nM unlabeled SST to determine the nonspecific binding. The incubation was carried out for 2 h at 22 C. Bound ligand was trapped by vacuum filtration through GF/C glass fiber filters (Whatman, Inc., Clifton, NJ) presoaked in 0.5% polyethilenimine. After three washes with 50 mM Tris, the filters were counted in a {gamma}-counter. SSTR densities and dissociation constants were found by nonlinear modeling, as previously reported (39). In this study, stable clones that expressed a similar receptor concentration were used (see Table 2Go).

Expression of mRNA for eNOS and nNOS
The identification of mRNA for both eNOS and nNOS in CHO-K1 cells was evaluated by means of RT-PCR experiments. Specific primers were designed for eNOS (sense, 5'-TGTGGCTGTCTGCATGG-3'; and antisense, 5'-TGGCTGGTAGCGGAAGG-3; GenBank accession no. AF110508) and nNOS (sense, 5'-GAATACCAGCCTGATCCA-3'; and antisense, 5'-CCAGGAGGGTGTCCACCGCA-3'; GenBank accession no. U67309). The amplification was carried out for 30 cycles at the following temperatures: 94 C (30"), 65 C (30"), and 72 C (30"). Expected length of the specific amplification products was 300 bp for eNOS and 700 bp for nNOS.

Determination of NO Production
Determination of Nitrate-Nitrite Accumulation.
NO produced from the conversion of L-arginine to L-citrulline is mainly oxidized to nitrite-nitrate, which is an indicator of NO synthesis. Nitrite-nitrate concentration was measured using the Nitrite/Nitrate Colorimetric Assay Kit (Cayman Chemical Co., Inc., Ann Arbor, MI). Briefly, cells were plated in 24-well plates at a density of 1 x 105/well; after 24 h they were treated with the test compounds for the indicated times; at the end of the incubation the cell culture medium was added to 10 µl of the Enzyme Cofactor mixture and to 10 µl of the Nitrate Reductase mixture following the manufacturer’s indications. OD (absorbance) was measured at 570 nm after 10 min. Nitrite/nitrate concentration was determined by a nitrate standard curve and calculated according to the kit instructions.

Determination of the [3H]-L-Citrulline Accumulation.
The conversion of L-arginine in L-citrulline was monitored measuring the production of [3H]-L-citrulline after incubation of the extract cytosolic fraction with [3H]-L-arginine, using the Stratagene kit, following the manufacturer’s indications. Briefly, cells are mechanically homogenized in a buffer containing 24 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1 mM EGTA, the particulate fraction is pelleted (14,000 x g, 5 min), and the cytosolic fraction is collected and incubated (10 mg/ml) in a reaction buffer (25 mM Tris HCl, pH 7.4; 1 µM flavin adenine dinucleotide; 1 µM flavin adenine mononucleotide; 3 µM tetrahydrobiopterin) in which 1.2 mM NAPDH, 0.25 µCi [3H]-L-arginine, and 750 µM CaCl2 have been added. The reaction was carried out for 60 min and then stopped with 400 µl 50 mM HEPES (pH 5.5)-5 mM EDTA. The formed [3H]citrulline, derived from the NO production, was then recovered by chromatography and measured in a ß-counter.

Immunoprecipitation
Cells were plated in 10-cm Petri dishes, until they reached 75% confluence. Then they were treated for the specified times with the test reagents. After two washes in PBS, cells were harvested in ice-cold lysis buffer containing 100 nM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40; 0.4 mM EDTA; 10 mM NaF; 2 mM vanadate; 10 mM Na pyrophosphate; 0.1 mg/ml phenylmethylsulfonyl fluoride; and a protease inhibitor cocktail (Cømplete, Roche). Soluble proteins (250 µg) were incubated for 2 h at 4 C with anti-eNOS or anti-nNOS antibody (1 µg/mg proteins) with continuous rotation. Then the samples were incubated for 1 h at 4 C, with continuous rotation, with antirabbit magnetic beads (Dynabeads, Dynal, Inc., Great Neck, NY) (3:1 vs. antibody) to separate the immunocomplexes.

Immunoblotting
Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.4; 140 mM NaCl; 2 mM EDTA; 2 mM EGTA; 10% glycerol; 1% Nonidet P-40; 1 mM dithiothreitol; 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride; and the Complete protease inhibitor cocktail (Roche). Proteins (25 µg) from each sample were size fractionated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Milano, Italy). Membranes were blocked with 25 mM Tris-HCl (pH 7.4), 140 mM NaCl, 2.5 mM KCl, and 0.1% Tween 20 containing 5% nonfat milk and probed with primary rabbit antibodies. The secondary antibody was a horseradish peroxidase-linked antirabbit IgG antiserum (Amersham). The antibody-reactive bands were visualized by enhanced chemiluminescence (Amersham).

[Ca++]i Measurement
Cells were plated on 25-mm glass coverslips and transferred to 35-mm Petri dishes. On the day of the experiment cells were washed for 10 min with a balanced salt solution (10 mM HEPES, pH 7.4; 150 mM NaCl; 5.5 mM KCl; 1.5 mM CaCl2; 1.2 mM MgSO4; 10 mM glucose). Then cells were loaded with Fura-2 penta-acetoxymethyl ester (4 µM) for 20 min at room temperature. Fluorescence measurements were performed as previously reported (5). Briefly, Fura-2 fluorescence was imaged with an inverted Nikon diaphot microscope using a Nikon 40x/1.3 NA Fluor DL objected lens (Nikon, Melville, NY). Fluorescence (ratio 340:380) was then evaluated and converted in intracellular Ca++ concentration ([Ca++]i) using the Quanticell apparatus (Visitech, Sunderland, UK). For the calibration of fluorescence signals, we used cells loaded with Fura-2; Rmax and Rmin are ratios at saturating and zero [Ca++]i, respectively, and were obtained perfusing the cells with a salt solution containing 10 mM CaCl2, 2.5 µM digitonin, and 2 µM ionomycin and subsequently with a Ca++-free salt solution containing 10 mM EGTA. The values of obtained Rmax and Rmin, expressed as gray mean level, were used to calculate the [Ca++]i, using Quanticell software (Visitech), according to the equation of Grynkiewicz et al. (43).

Statistics
All experiments were performed in triplicate. Statistical analysis was performed by means of one-way ANOVA.

P ≤ 0.05 was considered statistically significant.


    ACKNOWLEDGMENTS
 
We thank Dr. Michael Culler (Biomeasure, Inc., Milford, MA) for providing us with lanreotide and BIM23745and Dr. Susan Roher (Merck & Co., Inc., Rahway, NJ) for L-796778.


    FOOTNOTES
 
This work was supported by a grant from Italian Association for Cancer Research (AIRC 2003), and European Community contract QLG3-CT-1999–00908 (to G.S.).

First Published Online September 23, 2004

1 S.A. and A.P. contributed equally to this work and should both be considered first authors. Back

Abbreviations: BAPTA-AM, 1,2-Bis-(o-aminophenoxy) ethane- N,N,N',N'-tetracetic acid tetra(acetoxymethyl)ester; bFGF, basic fibroblast growth factor; [Ca++]i, intracellular Ca++ concentration; CCK, cholecystokinin; CHO, Chinese hamster ovary; eNOS, endothelial NOS; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, neuronal NOS; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PTX, pertussis toxin; SST, somatostatin; SSTR, SST receptor.

Received for publication July 9, 2004. Accepted for publication September 14, 2004.


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