Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production : key role of L-arginine availability.

BACKGROUND: Native and oxidized LDLs (n-LDL and ox-LDL) are involved in the atherogenic process and affect endothelium-dependent vascular tone through their interaction with nitric oxide (NO). METHODS AND RESULTS: In this study we evaluated directly, by using a porphyrinic microsensor, the effect of increasing lipoprotein concentrations on endothelial NO and superoxide (O(2)(-)) production. We investigated where lipoproteins may affect the L-arginine-NO pathway by pretreating cells with L-arginine, L-N-arginine methyl ester (L-NAME), and superoxide dismutase. Bovine aortic endothelial cells were exposed for 1 hour to increasing concentrations of n-LDL (from 0 to 240 mg cholesterol/dL) and ox-LDL (from 0 to 140 mg cholesterol/dL). A stimulated (calcium ionophore) NO concentration decreased to 29% of the control at n-LDL concentration of 80 mg cholesterol/dL and to 15% of the control at 20 mg cholesterol/dL of ox-LDL. L-Arginine partially neutralized the inhibitory effect of n-LDL and ox-LDL on the NO generation. Superoxide dismutase pretreatment did not modify NO production, whereas L-NAME blunted NO generation at all LDL concentrations. O(2)(-) production was increased at low n-LDL and very low ox-LDL concentrations; this was reversed by L-arginine. CONCLUSIONS: These findings confirm the inhibitory role of n-LDL and ox-LDL on NO generation and suggest that lipoproteins may induce a decreased uptake of L-arginine. The local depletion of the L-arginine substrate may derange the NO synthase, leading to overproduction of O(2)(-) from oxygen, the other substrate of NO synthase.

Glycosaminoglycans delay the progression of nephropathy in NIDDM.

OBJECTIVE: To determine the effect of oral administration of glycosaminoglycans on metabolic control and albumin excretion rate (AER) in NIDDM patients with increased urinary albumin excretion. RESEARCH DESIGN AND METHODS: Twelve NIDDM hypertensive patients (age 52 +/- 3 years, HbA1c 7.7 +/- 0.2%) on antihypertensive treatment were enrolled in a double-blind placebo-controlled study, assuming either placebo or sulodexide (100 mg/day) for 4 months; at the end of this period, a crossover was performed. We have evaluated routine biochemical parameters plus AER and coagulative function every 2 months. RESULTS: Both plasma fibrinogen (from 4.15 +/- 0.32 to 2.77 +/- 0.47 mmol/l) and AER (from 128.3 +/- 40.6 to 39.6 +/- 11.9 micrograms/min) decreased significantly after treatment with glycosaminoglycans in respect to placebo; moreover, blood pressure control ameliorated, also in the absence of any variation of therapy. CONCLUSIONS: Glycosaminoglycan therapy, likely in association with a satisfactory control of blood pressure values, seems to prevent the progression of diabetic nephropathy in NIDDM.

Stress-induced activation of sympathetic nervous system is attenuated by the delta-opioid receptor agonist deltorphin in healthy man.

To examine the role of delta-opioid receptors in the regulation of the sympathoadrenomedullary system, the effects of the highly selective delta-opioid receptor agonist deltorphin (DT) on plasma catecholamine responses to insulin-induced hypoglycemia (IIH) and cold pressor test (CPT) have been investigated in normal subjects in two separate studies. DT failed to modify basal plasma levels of both norepinephrine (NE) and epinephrine (E). DT completely suppressed the IIH-evoked elevation of NE, whereas it attenuated the E response by 20%, with the DT-induced decrease in E release failing to achieve statistical significance. DT completely blocked the release of both NE and E elicited by CPT. We conclude that specific delta-opioid receptor stimulation exerts an inhibitory effect on NE release induced by both IIH and CPT. These findings provide evidence that delta-opioid receptors may influence the autonomic sympathetic reactivity.

Evidence that enhancement of cholinergic tone increases basal plasma levels of calcitonin gene-related peptide in normal man.

To investigate the influence of the cholinergic system on the modulation of the circulating levels of calcitonin gene-related peptide (CGRP) under basal conditions in normal man, the effects of an acetylcholinesterase inhibitor, pyridostigmine bromide, and a muscarinic receptor blocker, pirenzepine, were studied in 16 normal subjects (8 females and 8 males). Pyridostigmine (120 mg, orally) induced a significant (P < 0.01) rise in basal plasma CGRP, while it reduced systolic and diastolic blood pressure. In all subjects, pirenzepine (0.6 mg/kg, i.v. bolus) was unable to modify the basal CGRP level. In conclusion, a pharmacologically induced enhancement of cholinergic tone resulted in an increase in CGRP, whereas muscarinic receptor blockade had no effect on CGRP levels or blood pressure. Therefore, the cholinergic system seems to be involved in the control of CGRP release in man, acting as a positive modulator. However, the available data do not indicate that there is a tonic cholinergic tone responsible for CGRP secretion under physiological conditions.

Progressive disruption of the circadian rhythm of melatonin in fatal familial insomnia.

Fatal familial insomnia (FFI) is a disease characterized by loss of sleep activity due to selective thalamic degeneration. To assess the secretory pattern of melatonin (MT) in FFI, we studied two cases of overt disease under standardized conditions and polysomnographic control. Each patient underwent repeated 24-h study sessions, and MT was assayed at 30-min intervals. Six healthy volunteers were used as controls. Slow wave sleep was never recorded, whereas occasional episodes of enacted dreaming accompanied by rapid ocular movements and complex muscular activities were documented, with no detectable rhythm. Plasma MT concentrations gradually decreased as the disease progressed. A significant circadian rhythm was detected in the earlier recordings, with decreasing amplitudes with disease progression. Complete rhythm obliteration was achieved in the most advanced stage. Normally placed nocturnal acrophases were detected in the earlier stages, but then a shift toward the daytime hours was observed. Thalamic lesions of FFI appear to determine a progressive disruption of the sleep/wake cycle accompanied by decreased circulating levels of MT, with progressive alterations in the circadian rhythm of this hormone. On the other hand, decreased secretion of MT may contribute to the sleep disturbances of FFI.

Diurnal blood pressure variation and hormonal correlates in fatal familial insomnia.

Fatal familial insomnia is a prion disease in which a selective thalamic degeneration leads to total sleep deprivation, hypertension, dysautonomia, adrenal overactivity, and impaired motor functions. With patients under continuous recumbency and polysomnographic control, we assessed the changes in the 24-hour patterns of blood pressure, heart rate, plasma catecholamines, corticotropin, and serum cortisol in three patients at different stages of the disease. Six healthy volunteers were used as control subjects. A dominant 24-hour component was detected at rhythm analysis of all variables, both in patients and control subjects. In the patients, the amplitudes gradually decreased as the disease progressed, leading to the obliteration of any significant dirunal variation only in the preterminal stage. A shift in phase corresponded to the loss of the nocturnal fall in blood pressure in an early stage of the disease, when nocturnal bradycardia was still preserved. Plasma cortisol was high and became increasingly elevated, whereas corticotropin remained within normal levels; abnormal nocturnal peaks appeared in their circadian patterns. The disrupted patterns of cortisol and blood pressure preceded the development of hypertension and severe dysautonomia, which in turn were paralleled by increasing catecholamine and heart rate levels. Our data demonstrate that in patients with fatal familial insomnia the changes detectable in the rhythmic component of diurnal blood pressure variability result in a pattern of secondary hypertension. Disturbances in thalamic, pituitary-adrenal, and autonomic functions seem to be involved in mediating these changes.

Modulatory effect of the renin-angiotensin system on the plasma levels of calcitonin gene-related peptide in normal man.

Calcitonin gene-related peptide (CGRP) has positive chronotropic and inotropic effects in animals and humans, and produces the most potent vasodilation known for an endogenous peptide. Yet, a physiological role for CGRP in the regulation of vascular tone and blood pressure has not been demonstrated. We studied the effects of 1) assumption of the upright position and 2) iv infusion of angiotensin-II (sequential doses of 8, 16, and 32 ng/kg.min, each dose for 20 min) in eight normal subjects (four men). Serial venous blood samples were taken to determine the plasma CGRP, epinephrine, norepinephrine, and aldosterone levels and PRA. Blood pressure and heart rate were continuously monitored at the finger with a Finapres 2300 instrument. After assumption of the upright posture, a quick rise in plasma CGRP levels was observed together with the expected increases in plasma norepinephrine and aldosterone and PRA. A transient increment was also observed for diastolic blood pressure and heart rate. Angiotensin-II infusion caused dose-dependent increases in plasma CGRP and aldosterone concentrations, already significant at the lowest infusion rate and parallel with the blood pressure rise. Plasma catecholamines significantly increased only at higher infusion rates. Our data demonstrate that modifications of plasma CGRP concentrations are part of the normal response to postural and vasomotor changes. These findings suggest a physiological role for CGRP in regulation of the peripheral vascular tone and possibly blood pressure in man.

Circadian rhythm of calcitonin gene-related peptide in uncomplicated essential hypertension.

OBJECTIVE: To assess the existence of an altered circulating pattern of calcitonin gene-related peptide (CGRP) in hypertension. DESIGN: The 24 h variation in plasma CGRP was measured and compared in 10 patients affected by uncomplicated essential hypertension and in nine age- and sex-matched healthy volunteers. The diurnal variations in blood pressure, atrial natriuretic peptide (ANP), plasma renin activity (PRA), plasma aldosterone and plasma cortisol were also assessed. METHODS: Recumbency studies were performed under standardized, drug-free conditions beginning at 0800 h. Venous samples were drawn every 4 h for 24 h and hormone levels were assessed with specific radioimmunoassays. The blood pressure was measured every 15 min with a SpaceLabs 90207 monitor. RESULTS: The mean 24-h plasma CGRP concentrations were significantly lower in the hypertensive group than in the control group. In both groups a circadian rhythm was present with the same pattern, but at a lower level in hypertension. A temporal sequence starting with the nocturnal rise in plasma CGRP concentrations and progressing with the elevations of ANP, PRA, and plasma aldosterone and cortisol was apparent in both groups. The nocturnal rise in the CGRP and ANP concentrations coincided with the blood pressure and the heart rate falls. CONCLUSIONS: Our data show that CGRP is lower than normal but maintains its circadian variability and its relationship with the diurnal variations in blood pressure and other hormones known to be active on the cardiovascular system.

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea.

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured. 2. ET-1 (1 pM-100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg. 3. The selective ET(B) receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM-100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET(A) receptor antagonist, FR139317 (10 microM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET(B) receptor antagonist, BQ788 (1 microM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively. 4. Exposure of tracheal segments to ET-1 (1-1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 microM) did not cause any significant NO release. FR139317 (10 microM), but not, BQ788 (1 microM), inhibited the NO release induced by ET-1. 5. These results demonstrate that in the isolated guinea-pig trachea activation of ET(B) receptors results in a contractile response, whereas activation of ET(A) receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

Time-dependent effect of isradipine on the nocturnal hypertension in chronic renal failure.

Nocturnal hypertension is frequently observed in chronic renal failure and contributes to the risk of target organ damages. We assessed whether antihypertensive therapy may restore a nocturnal blood pressure (BP) fall in this condition. A sustained-release oral formulation (SRO) of isradipine was used, and the possible differences in the response to morning nu evening dosing were also investigated. Sixteen hypertensive patients with chronic renal failure due to parenchymal kidney disease were studied after 2 weeks of single-blind placebo runin. According to the double-blind, randomized, cross-over design, they received 5 mg isradipine SRO at 08:00, or at 20:00 for 4 weeks, separated by a single-blind placebo period of 2 weeks. A 24-h BP monitoring at 10-min intervals was carried out at the end of each treatment using a SpaceLabs 90207 instrument. Under placebo, blunt BP profiles were observed, whereas HR showed a mean nocturnal fall of 17.4%, which remained unaltered after isradipine. Both isradipine treatments were equally effective in reducing the mean 24-h BP levels. However, the evening regimen showed a more pronounced effect during the night. The mean nocturnal fall in systolic/diastolic BP represented 4.8/8.7% and 7.5/10.9% of the corresponding daytime mean after morning and evening dosing, respectively. Only the evening administration reset the normal synchronization of the 24-h BP and HR profiles. Our findings demonstrate that antihypertensive treatment may restore a nocturnal BP fall in renal patients. An evening regimen of isradipine SRO seems more apt than a morning regimen to obtain this therapeutic goal.

Endocrine mechanisms of blood pressure rhythms.

The temporal organization of blood pressure is mainly controlled by neuroendocrine mechanisms. The monoaminergic systems appear to integrate the major driving factors of temporal variability, but evidence also indicates a role of the hypothalamic-pituitary-adrenal, hypothalamic-pituitary-thyroid, opioid, renin-angiotensin-aldosterone, and endothelial systems as well as other vasoactive peptides. Although their hormonal secretions are typically episodic, the probability of secretory episodes is "gated" by mechanisms that are coupled either to sleep or to an endogenous pacemaker which usually is predominantly (though not only) circadian. Many hormones with established actions on the cardiovascular system (arginine vasopressin, vasoactive intestinal peptide, melatonin, somatotropin, insulin, steroids, serotonin, CRF, ACTH, TRH, endogenous opioids, and prostaglandin E2) are also involved in sleep induction or arousal. Hence, physical, mental, and pathologic stimuli, which may drive activation or inhibition of these neuroendocrine effectors of biologic rhythmicity, may also interfere with the temporal blood pressure structure. On the other hand, the immediate adaptation of the exogenous components of blood pressure rhythms to the demands of the environment are modulated by the circadian-time-dependent responsiveness of the biologic oscillators and their neuroendocrine effectors. These notions may contribute to a better understanding of the pathophysiology and therapeutics of changes in blood pressure.

Fibrinolytic and coagulation factors in very old subjects: association with lipoprotein profile and anthropometric variables.

BACKGROUND: We evaluated plasminogen activator inhibitor-1 (PAI-1), factor VII activity (FVII), and fibrinogen in a sample of octo-nonagenarians. Furthermore, we investigated the relationship of these fibrinolytic and coagulation parameters with lipoprotein profile and anthropometric variables in the absence or presence of disability. METHODS: We enrolled a population of 162 octo-nonagenarians, divided in two groups on the basis of presence or absence of disability in the activity of daily living (ADL). All the anthropometric determinations were carried out according to standardized methods. Blood samples for hemostatic and lipid determinations were collected after overnight fasting and resting. RESULTS: PAI-1 activity and fibrinogen levels were significantly higher in disabled (DIS) compared to free-living (FL) adults, whereas FVII did not show differences in the two groups. PAI-1 activity and FVII positively correlated to anthropometric parameters (body mass index, subscapular and tricipital skinfold thickness) in both DIS and FL. No correlations were found between fibrinogen and other variables in FL, whereas a negative relation with high density lipoprotein-cholesterol levels emerged in DIS. FVII was positively related with total cholesterol low density lipoprotein-cholesterol, and apolipoprotein B in both FL and DIS. CONCLUSIONS: In a sample of octo-nonagenarians, PAI-1 activity and FVII show a significant correlation with several anthropometric and lipoprotein parameters, suggesting that these variables are strongly associated with body composition and lipid metabolism independent from age and disability. DIS presented higher PAI-1 and fibrinogen levels; this observation may take in account the high prevalence of vascular diseases and also occult inflammation, which are known to affect these parameters.

Relationships between fasting plasma insulin, anthropometrics, and metabolic parameters in a very old healthy population. Associazione Medica Sabin.

Several studies have shown that insulin resistance and hyperinsulinemia are associated with many metabolic disorders predisposing to coronary heart disease (CHD). This syndrome has been termed syndrome X. However, it is not completely known whether these relationships are still present in the elderly, or whether other factors such as age, gender, and body fat distribution modulate them. Therefore, we investigated the relationship between fasting plasma insulin, total and regional adiposity, fasting plasma glucose and lipids, plasma plasminogen activator inhibitor-1 (PAI-1), fibrinogen, and coagulation factor VII in a sample of 100 healthy free-living octogenarians-nonagenarians (52 men and 48 women) who were disability-free according to the Katz index. By univariate analysis, fasting insulin correlated positively with all anthropometric measures except the waist to hip ratio (WHR) in women. There was a positive correlation between fasting insulin and fasting glucose (r=.40, P < .01), plasma triglycerides ([TGs] r=.21, P < .05), and PAI-1 levels (r=.33, P < .01), whereas a negative relation was found with high-density lipoprotein cholesterol (HDL-C) and apolipoprotein, A-I (apo A-I) levels (r=-.22 and =-.24, respectively, P < .05). These relationships were weaker and less significant in women. In pooled data, stepwise multiple regression analysis showed an independent relationship of both the body mass index (BMI) and fasting insulin level with TGs (R2=.14), while gender and fasting insulin were the best predictors of HDL-C variance (R2=.17). Furthermore, fasting insulin was the only variable independently related to PAI-1 (R2=.12). Our findings support the existence of a metabolic syndrome even in very old age by showing that high insulin levels are related to various metabolic and hemostatic disorders.

Effect of dermorphin on behavior and hippocampal electrical activity in rabbits.

Dermorphin, a naturally occurring heptapeptide that selectively binds to mu-opioid receptors, was injected intravenously 0.4 mg/kg in male rabbits. Eight days before injection the spontaneous behavior of the animals was observed in a neutral environment in the absence of external stimuli. At the same time, hippocampal EEG was recorded by telemetry. After dermorphin injection, the same experimental procedure was repeated. Behavior showed a strong increase in quiet immobility and a concomitant reduction in voluntary activities as compared to control periods. Treatment did not affect either the pattern or the frequency of hippocampal electrical activity.

Effect of deltorphin on behavior and hippocampal electrical activity in rabbits.

To clarify the role of delta-opioid receptors on modulation of hippocampal electrical activity and behavior, deltorphin (DT), a naturally occurring heptapeptide that selectively binds to delta-opioid receptors, was intravenously (IV) administered to rabbits. For this purpose, at 8-day intervals, the effects of IV administration of normal saline and IV infusion of synthetic DT (1 mg/kg b.wt. for 2 min) on the spontaneous behavior in neutral environment, both in absence of any external stimulus and after the introduction of a stuffed predator, were examined in seven adult male rabbits, on separate and successive occasions. During each session of experimental procedure, hippocampal EEG was also recorded by telemetry. Behavioral activity showed an increase in alert and reactive immobility after the peptide injection in comparison with that observed during control period (saline administration). Under DT treatment the frequency of hippocampal electrical activity decreased, and a reduction in rhythmicity of electrical pattern was also observed in presence of stressful stimulus. These findings show that DT may affect neural and behavioral elements related to the control of attentional and emotional processes, suggesting a modulating role of delta-opioid receptors.

Atrial natriuretic peptide and circadian blood pressure regulation: clues from a chronobiological approach.

A critical review of the data available in the literature today permits a better understanding of the multiple actions of atrial natriuretic peptide (ANP) on the cardiovascular system. Moreover, the results of chronobiological studies suggest a role for this peptide in the determination of the circadian rhythm of blood pressure (BP). ANP can affect BP by several mechanisms, including modification of renal function and vascular tone, counteraction of the renin-angiotensin-aldosterone system, and action on brain regulatory sites. A series of interrelated events may follow from very small changes in the plasma levels of ANP. The endpoints are blood volume and BP reduction, but they are rapidly offset (mainly by reactive sympathetic activation) as soon as blood volume or pressure is threatened. The circadian rhythms of BP and ANP are antiphasic under normal conditions and in essential hypertension. The loss in the nocturnal decrease of BP is accompanied by a comparable loss in the nocturnal surge of ANP in hypertensive renal failure and hypotensive heart failure. In the latter condition, BP and ANP variabilities correlate significantly both before and after therapy-induced functional recovery, independently of the mean BP levels. Autonomic function modulates the secretion of ANP, which seems more apt to determine only transient changes in BP levels, as suggested by the short half-life of the peptide and the buffering role of its clearance receptors. There is now sufficient evidence that ANP contributes to short-term control over BP and electrolyte balance, in contrast and in opposition to the renin-angiotensin-aldosterone system, which is involved primarily in long-term BP control. By interfering with other well-established neurohormonal factors, ANP appears to be an additional modulator of the circadian rhythm of BP.

Consistent changes in the circadian rhythms of blood pressure and atrial natriuretic peptide in congestive heart failure.

We demonstrated in previous works that the circadian rhythms of blood pressure (BP) and atrial natriuretic peptide (ANP) are antiphasic in normal subjects and in essential hypertension. The aim of the present study was to assess the circadian rhythms of BP and ANP in 20 patients with stable congestive heart failure (CHF), divided into two groups of 10 according to their New York Heart Association functional class. A matched control group of 10 normal volunteers was also studied. Noninvasive BP monitoring at 15-min intervals was performed for 24 h. Peripheral blood samples were also obtained at 4-h intervals starting from 08:00 h. The mean (+/- SEM) circadian mesors of ANP plasma levels were 13.4 +/- 1.7 pmol/L in the control group, 28.6 +/- 2.4 pmol/L in the group of 10 patients in class II, and 81.5 +/- 12 pmol/L in the group of 10 patients in class III-IV. In normal subjects, plasma ANP concentration was highest at 04:00 h (21.5 +/- 2.7 pmol/L) and lowest at 16:00 h (8.8 +/- 2.4 pmol/L; p less than 0.01). Both groups of patients with CHF showed no significant circadian change in the plasma levels of ANP and also a significantly blunted circadian rhythm of BP. Cosinor analysis confirmed the loss of the circadian rhythms of ANP and BP in CHF patients. Our findings support the existence of a causal relationship between the circadian rhythms of ANP and BP.

Differences in blood pressure regulation of congestive heart failure, before and after treatment, correlate with changes in the circulating pattern of atrial natriuretic peptide.

The mechanisms underlying altered BP regulation in congestive heart failure are unknown. This study examines the possibility that differences in circadian blood pressure (BP) regulation between the normal and the failing heart correlate with changes in the circulating pattern of atrial natriuretic peptide (ANP). Twelve normotensive patients with coronary artery disease were studied over two separate 24-h periods, the first during acute exacerbation of congestive heart failure (radionuclide-determined ejection fraction at rest was less than 30%) and the second after therapy-induced functional recovery (ejection fraction was more than 40%). BP monitoring at 10-min intervals and intra-atrial blood samples for ANP assays at hourly intervals were obtained. Significant correlation between ejection fraction and the indexes of circadian BP variability (standard deviation of the 24-h pressure mean and day-night pressure difference) were found both before and after treatment. Ejection fraction was independent of the BP means (24-h, daytime and night-time). BP variability, 24-h mean and daytime mean were higher after treatment. ANP means were lower after treatment, whereas ANP variability was higher. The indexes of BP and ANP variability correlated both before and after treatment, whereas the BP and the ANP means were independent. These findings demonstrate that differences in BP regulation of CHF before and after effective treatment correlate with changes in the circulating pattern of ANP. We speculate that by modulating ANP release, the heart could be actively involved in BP regulation as part of the compensatory mechanisms aimed at protecting against circulatory overload.