Hemorrhagic shock : nitric oxide synthases activity and its association with caveolin-1 in rat heart

-.N. ARRECHE, L. SARATI, V. NETTI, A. FELLET, A.M. BALASZCZUK

Oslo

Congreso; 20 th European Meeting on Hypertension; 2010

Nitric oxide synthases (NOS) are involved in many aspects of cardiovascular homeostasis including regulation of blood pressure and heart rate (HR). Objectives: (i) to investigate the role of NO pathway in the cardiovascular adjustment in hemorrhaged rats (bleeding: 20% of the volemia) (ii) to study a relationship between caveolin-1 and NOS in this experimental model.(i) to investigate the role of NO pathway in the cardiovascular adjustment in hemorrhaged rats (bleeding: 20% of the volemia) (ii) to study a relationship between caveolin-1 and NOS in this experimental model. Methods: Groups of animals (n¼10/age group): S¼sham,H¼hemorrhaged rats, SþL-NAME (1 mg/kg iv and 0.5 mg/kg.h iv¼100fY ´ l/h), HþL-NAME. We evaluated in the left ventricle the NOS activity (NADPH-d technique), eNOS expression (Western blot) and coimmunoprecipitation analysis of caveolin-1 and eNOS (Immunofluorescence) at 60 and 120 min of bleeding. Analysis of variance (ANOVA) followed by the ad hoc Bonferroni test was used for multiple comparisons. The 5% probability level was used as a criterion for biological significance.Groups of animals (n¼10/age group): S¼sham,H¼hemorrhaged rats, SþL-NAME (1 mg/kg iv and 0.5 mg/kg.h iv¼100fY ´ l/h), HþL-NAME. We evaluated in the left ventricle the NOS activity (NADPH-d technique), eNOS expression (Western blot) and coimmunoprecipitation analysis of caveolin-1 and eNOS (Immunofluorescence) at 60 and 120 min of bleeding. Analysis of variance (ANOVA) followed by the ad hoc Bonferroni test was used for multiple comparisons. The 5% probability level was used as a criterion for biological significance.þL-NAME (1 mg/kg iv and 0.5 mg/kg.h iv¼100fY ´ l/h), HþL-NAME. We evaluated in the left ventricle the NOS activity (NADPH-d technique), eNOS expression (Western blot) and coimmunoprecipitation analysis of caveolin-1 and eNOS (Immunofluorescence) at 60 and 120 min of bleeding. Analysis of variance (ANOVA) followed by the ad hoc Bonferroni test was used for multiple comparisons. The 5% probability level was used as a criterion for biological significance. Results: L-NAME restored the hypotension induced by hemorrhage. Blood loss decreased heart rate in the first stage increasing at 60 and 120 min. LNAME blunted this effect. Left ventricle histochemical NOS activity increased at 60 and 120 min (21% and 45%, respectively). eNOS protein levels increased in left ventricle at 60 min without changes at 120 min compared with S group. eNOS exhibited a diffuse staining pattern throughout the ventricle cell and is significantly associated with caveolin-1 in S group. Meanwhile, at 60 min of bleeding a dissociation of caveolin-1 and eNOS was observed. The binding of caveolin-1 with eNOS is partially restored at 120 min. group. Meanwhile, at 60 min of bleeding a dissociation of caveolin-1 and eNOS was observed. The binding of caveolin-1 with eNOS is partially restored at 120 min. Conclusions: The involvement ofNO pathway on cardiovascular parameters occurs in an isoform-specific and time dependent manner after blood loss. This response would be influenced by direct protein interactions between the eNOS and it regulatory protein, caveolin-1 in hemorraghed rat.The involvement ofNO pathway on cardiovascular parameters occurs in an isoform-specific and time dependent manner after blood loss. This response would be influenced by direct protein interactions between the eNOS and it regulatory protein, caveolin-1 in hemorraghed rat.L-NAME restored the hypotension induced by hemorrhage. Blood loss decreased heart rate in the first stage increasing at 60 and 120 min. LNAME blunted this effect. Left ventricle histochemical NOS activity increased at 60 and 120 min (21% and 45%, respectively). eNOS protein levels increased in left ventricle at 60 min without changes at 120 min compared with S group. eNOS exhibited a diffuse staining pattern throughout the ventricle cell and is significantly associated with caveolin-1 in S group. Meanwhile, at 60 min of bleeding a dissociation of caveolin-1 and eNOS was observed. The binding of caveolin-1 with eNOS is partially restored at 120 min. group. Meanwhile, at 60 min of bleeding a dissociation of caveolin-1 and eNOS was observed. The binding of caveolin-1 with eNOS is partially restored at 120 min. Conclusions: The involvement ofNO pathway on cardiovascular parameters occurs in an isoform-specific and time dependent manner after blood loss. This response would be influenced by direct protein interactions between the eNOS and it regulatory protein, caveolin-1 in hemorraghed rat.The involvement ofNO pathway on cardiovascular parameters occurs in an isoform-specific and time dependent manner after blood loss. This response would be influenced by direct protein interactions between the eNOS and it regulatory protein, caveolin-1 in hemorraghed rat.