Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione.

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions. Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2'-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated. Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 µM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress. In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

[Pioglitazone protects against elastocalcinosis and improves aortic wall elasticity]. / Importance de l'activation des PPARgamma dans la pharmacologie de la rigidité artérielle.

Specific treatment of age-related aortic wall arteriosclerosis and stiffening is lacking. As anti-inflammatory ligands for peroxisome proliferator-activated receptor-gamma have beneficial effects on the arterial wall in atherosclerosis, we investigated whether chronic pioglitazone treatment protects against another form of vascular wall disease, arteriosclerosis. In a rat model of elastocalcinotic arteriosclerosis (hypervitaminosis D and nicotine, VDN), we evaluated whether pioglitazone attenuated arteriosclerosis and its consequences, aortic wall rigidity, increased aortic pulse pressure and left ventricular hypertrophy. In VDN rats, medial calcification was associated with monocyte/macrophage infiltration and induction of TNF-alpha and IL-1B. Pioglitazone decreased TNF-alpha and IL-1B mRNA expression, blunted aortic wall calcification and prevented fragmentation of elastic fibers. Pioglitazone reduced aortic wall stiffness, aortic pulse pressure and left ventricular hypertrophy. Our results may be clinically relevant in elderly patients suffering from aortic wall stiffening and isolated systolic hypertension.

Effect of chronic ANG I-converting enzyme inhibition on aging processes. IV. Cerebral blood flow regulation.

Age-related changes in systemic arterial blood pressure, basal cerebral blood flow (CBF), and CBF regulatory capacity were investigated in awake 6-, 12-, 24-, and 30-mo-old male Wistar (WAG/Rij) rats, one-half of which received the angiotensin I-converting enzyme inhibitor (ACEI) perindopril from 6 mo onward. There was no age-dependent change in mean arterial blood pressure, basal CBF, or cerebrovascular reactivity to hypercapnia, but the lower limit of CBF autoregulation rose from 70 mmHg at 6 and 12 mo to 90 mmHg in 24- and 30-mo-old animals. ACEI lowered mean arterial blood pressure but had no effect on basal CBF or on cerebrovascular reactivity to hypercapnia. ACEI shifted the lower limit of CBF autoregulation to a 20-mmHg-lower level in 12- and 24-mo animals but not in rats treated for 2 yr, i.e., from the ages of 6 to 30 mo. In conclusion, the main age-related change in CBF regulation was an increase in the lower limit of CBF autoregulation to a higher blood pressure level. Treatment with ACEI partially restored the lower limit of CBF autoregulation.

Vascular Ca overload produced by vitamin D3 plus nicotine diminishes arterial distensibility in rats.

In humans, aging produces many structural changes in blood vessels, one of the most pronounced being arterial calcium overload. Simultaneously arteries become increasingly rigid. The slow evolution of the two processes renders it difficult to evaluate the importance of vascular calcium overload in the development of decreased compliance. To gain insight into this relationship, rapid vascular calcium overload was produced by treating young rats with vitamin D3 and nicotine. When rats were allowed 16 days or longer to recover from such treatment, analysis of plasma parameters revealed no overt toxicity, and growth rate was similar to that of controls. Pronounced calcium overload was seen primarily in compliance arteries. Changes in systemic arterial compliance, characteristic impedance, pulse-wave velocity, and carotid compliance all reflected a substantial increase in arterial rigidity. Linear regression analysis revealed significant correlations between the various indicators of arterial distensibility and arterial calcium content. In conclusion, treatment of young rats with vitamin D3 and nicotine may provide a suitable model with which to investigate how calcium overload is involved in the induration of compliance arteries.

In vivo cerebrovascular reactivity in Wistar and Fischer 344 rat strains during aging.

Basal cerebral blood flow (CBF) and CBF regulation after hypercapnia and hypotensive hemorrhage were investigated using H2 clearance in the frontal cortex of awake 2-, 14-, or 23-mo-old Wistar or Fischer 344 rats. Basal CBF decreased in old Wistar but not in mature Wistar (old 64.4 +/- 2.8, mature 87.6 +/- 2.6, young 79.6 +/- 2.2 ml.min-1 x 100 g-1) or in old Fischer 344 (old 71.9 +/- 2.9, young 73.3 +/- 1.6 ml.min-1 x 100 g-1) rats. Cerebrovascular reactivity to hypercapnia decreased in mature and old Wistar (old 2.1 +/- 0.3, mature 3.1 +/- 0.7, young 7.0 +/- 2.1 ml.min-1 x 100 g-1 x mmHg-1) but not in old Fischer 344 rats (old 4.6 +/- 1.4, young 4.9 +/- 0.9 ml.min-1 x 100 g-1 x mmHg-1). The lower limit of CBF autoregulation increased by 20 mmHg during maturation and/or aging in the two strains. Because blood gases and pH evolved similarly in both strains, we postulate that differences in cerebrovascular structure and/or function explain the differences in CBF regulation in the older representatives of the two strains.

Chronic antihypertensive treatment with captopril plus hydrochlorothiazide improves aortic distensibility in the spontaneously hypertensive rat.

1. Adult male spontaneously hypertensive rats (SHR) were given captopril plus hydrochlorothiazide mixed in the diet for 10 weeks. Calculated daily doses were 44 mg kg-1 per day for captopril, and 22 mg kg-1 per day for hydrochlorothiazide. Separate groups received captopril or hydrochlorothiazide alone, at similar doses, or no treatment. A final group of WKY normotensive rats received no drug. 2. Systolic arterial blood pressure, measured at regular intervals throughout the 10 weeks' period was lowered but not normalized, in groups receiving either captopril plus hydrochlorothiazide, or captopril alone, but not in the group receiving hydrochlorothiazide alone. 3. Following pentobarbitone anaesthesia, systolic arterial blood pressure, measured in the femoral artery, was found to be lower in all treated groups, but the greatest effect was observed in SHR previously treated with captopril plus hydrochlorothiazide. Aortic pulse wave velocity was also lower in treated SHR, and once again the greatest decrease was observed in the group previously treated with captopril plus hydrochlorothiazide. 4. Following pithing, systolic arterial blood pressures were similar in all SHR groups. Aortic pulse wave velocity was lower in pithed rats previously treated with captopril and hydrochlorothiazide. 5. In conclusion, antihypertensive treatment of SHR produces falls in blood pressure and pulse wave velocity, an indicator of aortic distensibility. Results in pithed rats suggest that treatment with the combination of captopril plus hydrochlorothiazide may increase aortic distensibility independently of blood pressure.

Haemodynamic effects of the calcium facilitator Bay K-8644 in rats following vascular calcium overload.

1. The haemodynamic effects of the Ca2+ facilitator Bay K-8644 (Bay) were studied in a model of calcinosis induced by acute treatment with vitamin D3 and nicotine administration over 4 days with 13 days of recovery. 2. Calcium content of the left ventricular myocardium increased 8-9 fold, while aortic Ca2+ levels increased up to 12-fold in treated animals. There were minimal changes in the ECG and no change in the level of plasma alpha-hydroxy-butyrate-dehydrogenase, a cardiac specific enzyme which increases during ischaemia. Significant increases in pulse pressure (PP) were seen in anaesthetized and conscious calcinotic rats, with no increase in cardiac output index (DABF) or systemic vascular resistance. However, aortic rigidity (AORI) was significantly elevated in the calcinotic group under anaesthesia. 3. In both control and calcinotic rats, pressor responses to i.v. Bay were exclusively mediated by an increase in aortic blood flow (DABF) as lower body vascular resistance (TLBVR) did not change. The increase in DABF at low doses (0.1-1 microgram kg-1) of Bay probably resulted from an increase in venous return induced by the agonist, as Bay had little effect on cardiac contractility over this dose range (as estimated by left ventricular dp/dtmax) and did not cause tachycardia. At higher doses (10-1000 micrograms kg-1), Bay significantly increased LV dp/dt. Bay caused dose-related increases in AORI in pithed calcinotic rats, but a decrease in AORI in control animals. 4. The calcinosis model, which incorporates a recovery period to obviate the acute effects of nicotine and/or vitamin D3 treatment, results in long-term tissue calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cerebral blood flow. Changes in regulation with aging]. / Débit sanguin cérébral. Evolution de la régulation avec l'âge.

In subjects without cardiovascular or neuronal diseases ageing does not seem to be accompanied by a significant fall in global cerebral blood flow at rest. Yet non-negligible changes in cerebral blood flow rate can be demonstrated by haemodynamic or metabolic stimuli. Several intra- and extracranial vascular mechanisms may be involved in these changes. During ageing, a decrease of carotid artery compliance due to parietal calcium deposition might be an important factor in the disturbance of cerebral blood flow regulatory mechanisms.

Haemodynamic effects of a new dihydropyridine calcium entry blocker, S-12968-(-), in a rat model of cardiovascular calcium overload.

1. The haemodynamic effects of S-12968-(-), a new dihydropyridine calcium entry blocker (enantiomer of S-11568), were compared with those of the stereoisomer, S-12967-(+), nifedipine, and sodium nitroprusside. 2. A first experiment was performed in conscious, young male rats chronically implanted with femoral artery and vein cannula and repeated in rats previously treated with vitamin D3 and nicotine. Such treatment produces marked vascular calcium overload, especially of the compliance arteries, with no overt sign of toxicity as far as can be judged from the plasma profile. 3. In conscious rats the hypotensive effects of S-12968-(-), nifedipine and sodium nitroprusside were of similar potency. The falls in blood pressure produced by nifedipine and sodium nitroprusside were accompanied by reflex tachycardia which was less marked in the vascular calcium overload model. S-12968-(-) did not induce reflex tachycardia. S-12967-(+) increased blood pressure in both models. 4. A second experiment was performed in open-chest pentobarbitone-anaesthetized rats with electromagnetic flowprobes on the ascending aorta. In controls the falls in blood pressure produced by low doses (0.1 and 0.3 mg kg-1, i.v.) of S-12968-(-) were accompanied by falls in total peripheral resistance. The higher dose (1 mg kg-1, i.v.) of S-12968-(-) produced no change in total peripheral resistance, and in rats pretreated with vitamin D3 and nicotine, cardiac output fell. 5. In conclusion, S-12968-(-) appears to have a dual action and to lower blood pressure at higher doses at least in part by a cardiac effect. This phenomenon is more pronounced in rats pretreated with vitamin D3 and nicotine.6. S-12967-(+) resembles a calcium channel activator in this model.

Effects of the angiotensin I converting enzyme inhibitor perindopril on cerebral blood flow in awake hypertensive rats.

As chronic hypertension shifts the lower limit of cerebral blood flow (CBF) autoregulation to higher pressure levels, we studied the effects of the angiotensin converting enzyme (ACE) inhibitor, perindopril on mean arterial pressure (mean BP), basal CBF, and CBF autoregulation in awake renovascular hypertensive (2 kidneys, 1 clip model) and spontaneously hypertensive rats (SHR). Blood pressure was measured via a chronically implanted arterial cannula and CBF by hydrogen clearance. Chronic renovascular hypertension, like spontaneous hypertension, caused a marked shift in the lower limit of CBF autoregulation but did not alter basal CBF. In SHR, acute administration of perindopril did not diminish CBF in spite of the fact that BP fell to a level below the lower limit of CBF autoregulation (determined by hypotensive hemorrhage). Chronic treatment of renovascular hypertensive rats with perindopril normalized BP and restored CBF autoregulation.

Chronic treatment with the angiotensin I converting enzyme inhibitor, perindopril, restores the lower limit of autoregulation of cerebral blood flow in the awake renovascular hypertensive rat.

Chronic hypertension shifts the lower limit of cerebral blood flow autoregulation to a higher pressure level. Although acute administration of angiotensin converting enzyme inhibitors restores the lower limit of cerebral blood flow autoregulation the chronic effects have not received much attention. We studied the effect of the angiotensin converting enzyme inhibitor, perindopril, on mean arterial pressure, basal cerebral blood flow and cerebral blood flow autoregulation in renovascular hypertensive (two-kidney, one clip model) and normotensive male Wistar rats. Seven weeks after renal artery clipping or sham operation rats received daily intraperitoneal injections of perindopril. The dose was increased from 1 to 8 mg/kg over the first 4 weeks until blood pressure was normalized. Chronic renovascular hypertension caused a marked shift in the lower limit of cerebral blood flow autoregulation but did not alter basal cerebral blood flow. Treatment of hypertensive rats with perindopril normalized blood pressure and restored cerebral blood flow autoregulation. Chronic treatment of normotensive rats with perindopril increased basal cerebral blood flow. In conclusion, chronic treatment of renovascular hypertensive rats with perindopril causes a shift in the lower limit of cerebral blood flow autoregulation towards the value observed in normotensive rats.

Morphological and neurochemical effects of diazepam and phenobarbital on selective culture of neurons from fetal rat brain.

The responses to diazepam (DZ) and phenobarbital (PhB) were studied in enriched neuronal primary cultures from rat embryo hemispheres. Cells were grown in chemically defined medium and the drugs were added for 3 days to cultures, at pharmacologically active concentrations. Following exposure to DZ or to PhB, morphological changes, such as less prominent neuronal processes, were observed in neurons. It was also shown that each drug reduced the specific uptake of 2-deoxy-D-glucose by the cells and interfered with protein and RNA metabolism. It was concluded that both DZ and PhB might affect, at least transiently, the normal growth of neurons in culture.