Structure-activity studies on bradykinin and related peptides: agonists.

1. Bradykinin, kallidin, T-kinin, [Hyp3]-bradykinin and several analogues were prepared by solid-phase synthesis and purified by high performance liquid chromatography. 2. The various peptides were tested for their abilities to relax the dog carotid and renal arteries, or to contract the rabbit jugular vein and aorta, in order to measure their activities on BK2 (the first three preparations) or BK1 (the rabbit aorta) receptors. The dog renal artery without endothelium was also used as a BK1 receptor system. 3. T-kinin was found to be less active than bradykinin, while the replacement of Pro3 with Hyp favoured BK2 receptor occupation. [Hyp3,Tyr(Me)8]-BK was found to be a selective BK2 receptor agonist. 4. Amidation or methylation of the C-terminal carboxyl decreased activity, while extension of the N-terminal with Sar or D-Arg increased affinity and selectivity for BK1 (Sar) and affinity for BK2 (D-Arg) receptors. Acetylation of N-terminal amide brought affinity down to 10% or less. 5. Replacement of the peptide bonds Phe8-Arg9 to protect from kininase I and II, decreased affinities slightly, but was incompatible with additional changes at the N-terminal or in the peptide bond Gly4-Phe5. 6. Substitution of C-terminal Phe in desArg9-BK (the BK1 receptor stimulant) with D-Phe increased potency and selectivity for BK1 receptors while protecting from carboxypeptidases. Sar[D-Phe8]desArg9-BK was found to be a potent and selective BK1 receptor agonist.     

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c ^{+ /hi} B220^? DCs isolated from spleen and Peyer's patches (PP) of cow's milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE‐ and IgG‐specific responses were evaluated. Experiments were also carried out to determine the levels of interferon‐γ (IFN‐γ) and interleukin (IL)‐4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)‐mediated IL‐10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM‐specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL‐10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen‐specific IgE responses and that a Th2‐skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.     

Seasonal variation of trace metal concentrations in the digestive gland of the Mediterranean mussel Mytilus galloprovincialis: Comparison between a polluted and a non-polluted site

Seasonal changes in metal (Cu, Fe, Mn, Pb, and Zn) concentrations were observed in the digestive gland of the Mediterranean mussel, Mytilus galloprovincialis, from both a polluted and a non-polluted population. Digestive gland of mussels from the polluted site showed metal concentrations appreciably higher than in non-polluted organisms, especially for Pb (up to 160 g/g d.w.), Mn (up to 300 g/g d.w.), and Fe (up to 8,500 g/g d.w.), whereas the two populations showed maximum mean values, respectively, of 34 and 20 g/g d.w. for Cu and 170 and 120 g/g d.w. for Zn.Over 1 year (1991), differences between maximum and minimum values were moderate only in the unpolluted organisms. During gametogenesis, while the metal concentrations (expressed as g/g dry weight) decreased in the digestive gland, the tissue burden (as g) in that organ remained nearly constant or increased slightly.The apparent decrease in metal concentrations was probably due to the penetration of gonadic tissues into the digestive gland during gametogenesis, which biologically diluted metal concentrations in mussels from both the polluted and unpolluted populations. The effect of mussel size on trace metal concentrations in the digestive gland was demonstrated during different stages of the reproductive cycle. Higher concentrations were found in smaller organisms; however, during the spawning period, due to the fact that this is not a synchronous process within a population, the organisms exhibit a high variability in digestive gland weight which can mask this relationship when digestive gland weight is used as a size-index and as a parameter of the trace metal digestive gland burden.     

Effects of intrathecal nocistatin on the flexor reflex and its interaction with orphanin FQ nociceptin

We studied the effects of intrathecal (i.t.) nocistatin, a peptide identified from the precursor of orphanin FQ/nociceptin (OFQ) on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats and its interaction with i.t. OFQ. Nocistatin induced a moderate, non-dose-dependent facilitation of the flexor reflex without producing reflex depression whereas i.t. OFQ induced a biphasic dose-dependent facilitatory and inhibitory effect. The facilitatory effect of low dose (0.55 pmol) OFQ was significantly increased by nocistatin. On the other hand, the duration, but not magnitude, of reflex depression induced by a high (550 pmol) dose of OFQ was significantly shortened by 5.5 nmol nocistatin. Thus, nocistatin interacts with OFQ in a complex fashion, increasing excitation and reducing inhibition. No evidence was obtained for an antinociceptive effect of nocistatin in rat spinal cord.     

Effect of nociceptin on alcohol intake in alcohol-preferring rats

The present study investigated the effect of nociceptin (NC), the endogenous ligand of the opioid-like orphan receptor ORL1, on ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. Acute intracerebroventricular (ICV) injection of 250 or 500 ng/rat of NC, just before access to 10% ethanol (offered 2 h/day), significantly increased ethanol intake. Subchronic (7 days) ICV injection of 500 ng/rat of NC, given just before access to 10% ethanol (for 30 min/day), resulted in a progressive decrease in ethanol consumption. After the end of NC treatment, rats progressively recovered their usual ethanol intake. When NC, 500 or 1000 ng/rat, was tested versus the effect of ethanol in the place conditioning paradigm, NC significantly reduced the increase in time spent in the ethanol-paired compartment after conditioning. This finding suggests that NC reduces the rewarding properties of ethanol in msP rats; thus, they may respond to the acute NC administration by increasing their ethanol intake in an attempt to achieve the usual reinforcing effect of ethanol, whereas subchronic NC treatment may result in extinction of ethanol drinking. The results of the present study suggest that the brain NC mechanisms may represent an interesting target of pharmacological interventions for the treatment of alcoholism. Received: 11 August 1998/Final version: 15 October 1998     

A new bioassay for glucagon

1 The relaxant action of glucagon has been studied in strips of rabbit renal arteries partially contracted by a low concentration (1 ng/ml) of noradrenaline.

2 The preparation was relaxed in a dose-dependent manner by concentrations of glucagon varying between 25 ng/ml and 420 ng/ml.

3 The relaxant effect of glucagon (0.1 μg/ml ED₆₀) on this preparation was not affected by propranolol (5.0 μg/ml), cimetidine (10 μg/ml), diphenhydramine (10 μg/ml), indomethacin (5.0 μg/ml), phentolamine (1.2 μg/ml), atropine (10 μg/ml) and 8-Leu-AT_II (1.0 μg/ml) but was slightly potentiated by Des-Arg⁹ Leu-OMe⁸-Bk (25 μg/ml) and indomethacin (50 μg/ml).

4 The dose-response curve to glucagon remained parallel in the presence of papaverine (2.5 μg/ml) but was shifted to the left by a factor of 2.5 to 2.8. Theophylline (250 μg/ml) also potentiated the vascular relaxation induced by glucagon.

5 Insulin (10 μg/ml) did not influence the relaxant effect of glucagon.

6 The removal of the N-terminal amino acid (His) of glucagon reduced by 89% the biological activity of this fragment on the vascular preparation. The removal of the C-terminal amino acids Met-27, Asn-28 and Thr-29 of glucagon resulted in a fragment which was inactive either as an agonist or as an antagonist when tested at concentrations as high as 925 ng/ml.

7 It is concluded that the relaxation of partially contracted strips of rabbit renal arteries by glucagon constitutes a simple, sensitive, relatively specific and reliable bioassay which may be useful for the determination of glucagon in biological materials and for structure-activity relationship studies with this hormone.

     

Long-term outcome in diabetic heart failure patients treated with cardiac resynchronization therapy

BACKGROUND: Diabetes mellitus is an independent risk factor for increased morbidity and mortality in heart failure (HF) patients. AIMS: To compare functional and structural improvement, as well as long-term outcome, between diabetic and non-diabetic HF patients treated with cardiac resynchronization therapy (CRT). METHODS: We compared response to CRT in 141 diabetic and 214 non-diabetic consecutive patients. Major events were; death from any cause, urgent heart transplantation and implantation of a left ventricular (LV) assist device. Frequencies of hospitalisation and defibrillator (CRT-D) discharges were also analyzed. RESULTS: CRT was able to significantly improve functional capacity, ventricular geometry and neurohumoral imbalance in both diabetic and non-diabetic patients over a median follow-up time of 34 months. Overall event-free survival was similar in diabetic and non-diabetic patients (HR 1.23, p=0.363), as was survival free from CRT-D interventions (HR 1.72; p=0.115) and hospitalisations (HR 1.12; p=0.500). On multivariable analysis, NYHA class IV (p=0.002), low LV ejection fraction (p=0.002), absence of beta-blocker therapy (p<0.001), impaired renal function (p=0.003), presence of an epicardial lead (p=0.025), but not diabetes (p=0.821) were associated with a poor outcome after CRT. CONCLUSIONS: Diabetic HF patients treated with CRT had a very favourable functional and survival outcome, which was comparable to non-diabetic patients.     

A comparative study of the in vitro antioxidant activity of statins

Background: Treatment of hypercholesterolemia with statins is remarkably effective in cardiovascular prevention. This has led to the hypothesis that these drugs may act on the atherosclerotic plaque by mechanism(s) independent of the reduction of serum cholesterol levels. The aim of this study was to assess the total antioxidant activity of the most prescribed statins: fluvastatin, atorvastatin, pravastatin and simvastatin. Methods: We measured the in vitro antioxidant activity of statins as their ability to antagonize the oxidation of -keto-γ-methiolbutyric acid by both hydroxyl and peroxyl radicals. The results are expressed as Total Oxyradical Scavenging Capacity (TOSC) units. Uric acid and Trolox were used as the reference antioxidants. Results: The scavenging capacity towards hydroxyl radicals was highest for simvastatin (3375±112 U/mg), a value 270.2% higher (P<0.0001) compared to uric acid (reference antioxidant vs. hydroxyl radicals, 1249±71 U/mg). Among the tested statins, fluvastatin exhibited the highest anti-peroxyl radical antioxidant capacity (8755±187 U/mg) which appeared 50% lower (P<0.0001) compared to Trolox (reference antioxidant vs. peroxyl radicals, 17 460±379 U/mg). Conclusions: All the statins tested have intrinsic antioxidant activity with both anti-hydroxyl and peroxyl radical activity. Simvastatin was the most effective as an anti-hydroxyl radical antioxidant and fluvastatin as an anti-peroxyl radical antioxidant.     

Characterization of pre- and postjunctional receptors for neurokinins and kinins in the rat vas deferens

Neurokinins and kinins are potent stimulants of the rat vas deferens: they increase both the twitch response to electrical stimulation by facilitating transmitter release and the basal tone of the preparation by activating smooth muscle receptors. The two sites of action have been studied separately in the present experiments by using the prostatic for the prejunction site and the epididymal section of the vas deferens for the postjunction site. Receptors for neurokinins, characterized by the order of potency of agonists, appear to be of the NK-A type both at the pre- and postjunction level. Receptors for kinins are present also at both sites and are probably of the B2 type: they have been characterized by the use of agonists and, the B2 postjunction type has been convalidated with antagonists. These compounds could not be used for the prejunction receptor characterization, because they act as agonists. Receptor for angiotensin at the prejunction level are of the same type as in the cardiovascular and other systems.