Does polyphasic fever-inducing dose of lipopolysaccharide (LPS) modulate expression of inducible isoforms of NOS and HO in the rat locus coeruleus whose activities can be altered respectively by ZnDPBG and L-NMMA?

RN Soriano1, M Kwiatkoski3, GR Oliveira-Pelegrin2, AS Soares2, ME Batalhao1, MJ Rocha2, LGS Branco2, EC Carnio1. 1University of Sao Paulo, General and Specialized Nursing (DEGE), EERP - 14040902, Brazil, 2University of Sao Paulo, MEF - Dental School, FORP - 14040904, Brazil, 3University of Sao Paulo, Medical School, FMRP - 14049-900, Brazil

The rat locus coeruleus (LC) participates in fever. Despite both ZnDPBG [zinc(II)deuteroporphyrin IX 2,4 bis ethyleneglycol] and L-NMMA (NG-monomethyl-L-arginine acetate) potentially diminish the carbon monoxide (CO) and nitric oxide (NO)-mediated soluble guanylate cyclase (sGC) stimulation, ZnDPBG within the LC exerts a propyretic effect whereas L-NMMA is antipyretic. We hypothesized that expression of inducible isoforms of LC NO synthase (NOS) and heme oxygenase (HO) are increased during fever. Aiming at further exploring the mechanisms underlying these pro- and antipyretic properties, we investigated the putative interplay between the pathways inhibited by ZnDPBG and L-NMMA in the LC and whether inducible isoforms of NOS and HO are involved. Under general anesthesia (ketamine-xylazine, 100 and 10 mg/kg, intraperitoneal), male Wistar rats (300-340g) were implanted with a guide cannula toward the fourth ventricle (4V). Experiments started 08-0900AM, at 23°C, one week after the surgery. Awake, freely moving rats were bolus microinjected intra-4V with the HO inhibitor (ZnDPBG; 75 nmol/2 μl), or a CO donor (CORM-2, [tricarbonyldichlororuthenium-(II)-dimer]; 50 µmol/2 µl), or the NOS inhibitor (L-NMMA; 4 μmol/2 μl), or a NO donor (NOC12, [3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene]; 100 nmol/2µl), and bolus injected with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneal). L-NMMA and NOC12 were dissolved in saline, CORM-2 in 0.5% dimethylsulfoxide, and ZnDPBG in Na2CO3 (50 mM). Two or four hours after microinjection+LPS or only LPS, the rats were decapitated, and their brains promptly frozen and cut in a cryostat. LC punches (500 µm-thick, 500 µm-radius) were excised (landmarks: tract of the facial nerve and the 4V) and sonicated either in sterile buffered saline (60 µl) to assess the levels of bilirubin (with a commercial kit) and nitrite/nitrate (NOx; with a device, NO Analyzer) or in a buffer (30 µl; 1% Triton X-100/1% sodium deoxycholate/0.1% NaDodSO4/150 mM NaCl/10 mM Tris HCl, pH 7.2) to evaluate, by Western blot, expression of inducible (i)NOS and HO-1 (inducible). Procedures approved by local Ethics committee (10.1.305.53.9). Data are expressed as mean±standard error. Statistical analyses: one-way ANOVA followed by Tukey’s post hoc test. In febrile rats, microinjection of ZnDPBG reduced LC bilirubin (0.227±0.032 vs. 0.100±0.022 mg of bilirubin/mg of protein; P<0.05; n=9-11) and increased LC NOx (225.26±26.49 vs. 395.69±44.61 pmol of NOx/mg of protein; P<0.05; n=9-12), whereas L-NMMA diminished LC NOx (225.26±26.49 vs. 71.77±16.18; P<0.05; n=9-12) and reduced LC bilirubin (0.227±0.032 vs. 0.112±0.029; P<0.05; n=9-11); NOC12 tended to augment LC bilirubin (0.227±0.032 vs. 0.261±0.036; P>0.05; n=9-11), whereas CORM-2 tended to diminish LC NOx (225.26±26.49 vs.103.75±27.04; P>0.05; n=9-12). LPS did not alter expression of either LC iNOS (1.01±0.39 vs. 0.47±0.19; P=0.42; n=4-7) or LC HO-1 (0.41±0.07 vs. 0.60±0.13; P=0.18; n=4-8). These data suggest that polyphasic fever-inducing dose of LPS does not seem to increase expression of iNOS and HO-1 in the LC, revealing that increases in NOS and HO activities are not associated with changes in expression, and that an interplay exists between NOS and HO. Financial support: FAPESP.