Inflamm. res. 56, Supplement 1 (2007) S39–S40 1023-3830/07/01S41-02 DOI 10.1007/s00011-006-0519-5 Inflammation Research © Birkhäuser Verlag, Basel, 2007 Nitric oxide involvement in histamine-mediated PANC-1 cells growth G. Cricco 1 , V. Medina 1 , M. Núñez 1 , N. Mohamad 1 , A. Gutiérrez 1 , R. Bergoc 1,2 , E. Rivera 1 and G. Martín 1,2 1 Radioisotopes Laboratory, School of Pharmacy and Biochemistry. University of Buenos Aires. Junín 956 PB (C1113AAB), Buenos Aires, Argentina, Fax: ++54 1149648202, e-mail: gamartin@ffyb.uba.ar 2 National Research Council (CONICET), Argentina Published Online First 14 March 2007 Introduction Nitric oxide (NO) is a pleiotropic, ubiquitous modulator of many cellular functions, synthesized from L-arginine by ni- tric oxide synthases (NOS). The role of NO in tumor biology is controversial [1]. It is known that histamine (HA) induces NOS expression and increases NO production via H 1 hista- mine receptor (H 1 R) in endothelial cells [2]. The human pancreatic carcinoma cells PANC-1 over- express H 1 R and H 2 R. HA concentrations higher than 1 μM inhibit cell proliferation while low doses of HA (10 nM) stimulate cell growth. In these cells H 1 R are coupled to PLC signaling pathway and H 2 R are associated with both PLC and cAMP metabolic pathways [3]. The aim of this work was to study HA-induced NOS modulation and its possible involvement in PANC-1 cell growth. Materials and methods Cell Culture PANC-1 cells (ATCC CRL 1649) were cultured in 10 % FBS- RPMI medium at 37 °C in a humidified atmosphere. Detection of intracellular NO levels 5 × 10 5 cells were cultivated with 10 μM HA, 10 μM dimaprit dihydro- chloride (H 2 agonist), 10 μM 2-(3-(trifluoromethyl) phenyl)histamine (H 1 agonist), 10 μM 3-morpholisnosydnonimine (SIN-1), 300 μM aminoguanidine (AG), 4 mM L-NAME or were left without treatment (Control). At 24 h cells were washed with PBS, and incubated with the fluorescent dye DAF-2DA (5 μM) in PBS containing 1 mM L-arginine at 37 °C. After 30 min cells were washed and harvested to be analyzed on a FACScalibur flow cytometer (Becton Dickinson) employing WindMI 2.8 software (Scripps Institute, La Jolla, CA). Evaluation of NOS isoforms by RT-PCR Expression of inducible and endotelial NOS (iNOS and eNOS) was studied after 24 h of treatment. Total RNA was isolated using Trizol®. The MMLV-RT enzyme was employed for retrotranscription. The result- ing cDNA was amplified by PCR in the presence of each specific primer (sense and antisense), dNTPs and Taq polymerase in a thermocycler (Perkin Elmer, GeneAmp PCR System 2400), in time and temperature conditions as appropriate [4]. Results and discussion In this work using RT-PCR, we determined that PANC-1 cells only express constitutive eNOS isoform. In Figure 1A the eNOS mRNA expression was induced when cells were incubated with high doses of HA or forskolin (Fk: a direct activator of adenylate cyclase), suggesting a potential role of cAMP-dependent protein kinase (PKA) in eNOS activation similar to that reported in platelets [5]. It has been demonstrated that multiple protein kinases like Akt/PKB, PKA, PKC, CaMK II and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to different stimuli [6]. Recently, HA-modulation of eNOS transcription via H 1 R by a signaling pathway involving CaMK II has been described in endothelials cells [2]. Our results also indicated that the NO donor SIN-1 down-modulated the steady-state of eNOS mRNA showing a negative feedback on eNOS mRNA expression. However, AG (a NOS inhibitor) produced a positive modulation of eNOS level at 24 h. This discrepancy might be explained as a molecular mechanism employed by PANC-1 cells to bypass the enzymatic inhibition and supports the idea that a narrow range of NO intracellular concentration is required for cell growth. It has been published that NO produced in tumors can either positively or negatively regulate growth, depending on defined threshold concentrations of NO as well as duration of exposure in order to modify the response of key signal transduction proteins [7]. In this sense our proliferative assays indicated that NOS inhibitors exerted a dose-dependent inhibitory effect in cell proliferation. Correspondence to: G. Martín