Cigarette smoke-induced alterations in endothelial nitric oxide synthase phosphorylation: role of protein kinase C.

Endothelial nitric oxide synthase (eNOS) is regulated by phosphorylation of Ser(1177) and Thr(495), which affects NO bioavailability. Cigarette smoke disturbs the eNOS-cGMP-NO pathway and causes decreased NO production. Here the authors investigated the acute effects of cigarette smoke on eNOS phosphorylation, focusing on protein kinases (PKs). Endothelial cell culture was concentration- and time-dependently treated first with cigarette smoke buffer (CSB), then with reduced glutathione (GSH) or various PK inhibitors (H-89, LY-294002, Ro-318425, and ruboxistaurin). eNOS, phospho-Ser(1177)-eNOS, phospho-Thr(495)-eNOS, Akt(PKB), and phospho-Akt protein levels were determined by Western blot. CSB increased the phosphorylation of eNOS at Ser(1177) and more at Thr(495) in a concentration- and time-dependent manner (p < .01, p < .05 versus control, respectively) and resulted in the dissociation of the active dimeric form of eNOS (p < .05). GSH decreased the phosphorylation of eNOS at both sites (p < .05 versus CSB without GSH) and prevented the decrease of dimer eNOS level. CSB treatment also decreased the level of phospho-Ser(473)-Akt (p < .05 versus control). Inhibition of PKA by H-89 did not affect CSB-induced phosphorylation, whereas the PKB inhibitor LY-294002 enhanced it at Ser(1117). The PKC blockers Ro-318425 and ruboxistaurin augmented the CSB-induced phosphorylation at Ser(1177) but decreased phosphorylation at Thr(495) (p < .05 versus CSB). Cigarette smoke causes a disruption of the enzymatically active eNOS dimers and shifts the eNOS phosphorylation to an inhibitory state. Both effects might lead to reduced NO bioavailability. The shift of the eNOS phosphorylation pattern to an inhibitory state seems to be independent of the PKA and phosphoinositol 3-kinase (PI3-K)/Akt pathways, whereas PKC appears to play a key role.

Single dose of acetylsalicylic acid in patients with Type 2 diabetes mellitus and/or chronic renal failure ameliorates anaemia by decreasing the rate of neocytolysis.

BACKGROUND: Anaemia in diabetes mellitus (DM) and/or chronic renal failure (CRF) may be caused by a decreased production of erythropoietin (EPO), EPO resistance, and by the lysis of the young circulating red blood cells (neocytolysis) induced by subclinical inflammation and low EPO level. Aims of this study were to detect EPO resistance in patients with DM and/or CRF and to prove, that acetylsalicylic acid (ASA) is able to improve the haemopoietic status by decreasing neocytolysis. METHODS: In a cross-sectional study, three groups of selected patients (patients with DM; patients with DM+CRF; patients with CRF without DM, n=15 each) and a group of controls (non-diabetic, nonazotemic subjects, n = 10) were compared. In the intervention part of the study, the effect of a single dose of 1 gram ASA on neocytolysis was investigated in a subgroup of these patients. RESULTS: Despite the similar EPO level (p = 1.000), all three patient groups had lower haemoglobin and haematocrit than control persons (p < 0.05 in all cases). Patients with DM+CRF had lower haemoglobin than patients with DM or CRF alone (p < 0.05). Single dose of ASA induced a fast increase in serum EPO level, a concomitant rise of the Rtc number and rate, red blood cell count, haematocrit and haemoglobin p < 0.01 for each). These changes were accompanied by a marked decrease in serum lactate dehydrogenase activity (p < 0.01). CONCLUSIONS: DM and CRF may induce erythropoietin resistance. In these patients, ASA treatment increases serum EPO level. The higher EPO level and the anti-inflammatory effect of ASA may decrease neocytolysis.