Increased levels of plasma neudesin in adult growth hormone deficiency and their relationship with plasma liver-expressed antimicrobial peptide-2 levels: a cross-sectional study.

PURPOSE: Adult growth hormone deficiency (aGHD) is characterized by an altered metabolic profile and increased cardiovascular risk. Neudesin is a newly discovered protein mainly secreted from adipose tissue and brain, under evaluation for its possible activity as a negative regulator of energy expenditure. Liver-expressed antimicrobial peptide (LEAP)-2 is a competitive antagonist of ghrelin on its receptor. An observational cross-sectional study was performed to test the hypothesis that plasma neudesin levels may be modified in aGHD. Given the role played in the energy balance, any possible relationships between neudesin, LEAP-2 and metabolic and anthropometric parameters were evaluated. SUBJECTS AND METHODS: Thirty-eight patients were included: 18 aGHD patients (7 females and 11 males, aged 59.7 ± 2.6 years, BMI 30.2 ± 2.2 kg/m2); 20 healthy controls (12 females and 8 males, aged 47.1 ± 2.5 years, BMI 24.1 ± 0.9 kg/m2). All patients were evaluated for glucose, insulin, HOMA and QUICKI index, total/LDL/HDL cholesterol, triglycerides, uric acid, and IGF-1. Plasma neudesin, LEAP-2, and ghrelin were measured by ELISA. Fat mass was evaluated by DEXA. RESULTS: Neudesin levels were significantly higher in aGHD versus controls. We confirmed the finding of significantly lower ghrelin levels and significantly higher LEAP-2/ghrelin ratio in aGHD patients and found a significant direct correlation between neudesin and LEAP-2 levels. A significant direct correlation between neudesin and fat mass percentage was found in the whole population. CONCLUSION: These results suggest the onset of adaptive responses to an altered metabolic picture in aGHD. The changes in two distinct pathways that modulate food intake and the still limited knowledge about neudesin suggest future developments in this field.

Aggressive multiple sclerosis: a single-centre, real-world treatment experience with autologous haematopoietic stem cell transplantation and alemtuzumab.

BACKGROUND AND PURPOSE: The best therapeutic approach for aggressive relapsing-remitting multiple sclerosis remains unknown. The objective was to compare the efficacy and safety of autologous haematopoietic stem cell transplantation (aHSCT) and alemtuzumab in aggressive relapsing-remitting multiple sclerosis. METHODS: The time to first relapse, time to confirmed disability worsening, time to first evidence of magnetic resonance imaging (MRI) activity and time to first evidence of disease activity were compared between the two treatment groups. Secondary outcomes included the 12, 24 and 36 month annualized relapse rate (ARR) and the 6-month confirmed Expanded Disability Status Scale (EDSS) changes at months 12 and 24. RESULTS: Fifty-seven patients treated with aHSCT (n = 25) or alemtuzumab (n = 32) were included. At baseline, aHSCT patients had a higher EDSS (median score 6 vs. 3; P < 0.001), higher ARR (mean ARR 3.2 vs. 1.7; P = 0.001) and a higher number of baseline T1 gadolinium-enhancing lesions on MRI (mean number 15.5 vs. 1.6; P < 0.001). NEDA-3 (no evidence of disease activity) status was more frequently achieved in aHSCT-treated patients than in alemtuzumab-treated patients [75% vs. 56% of patients at the end of the observation period; hazard ratio (HR) 0.27, 95% confidence interval (CI) 0.08-0.84; P = 0.023]. aHSCT significantly reduced the risk of relapse (relapse-free survival 84% vs. 69%; HR 0.13, 95% CI 0.02-0.63; P = 0.012) and MRI activity (MRI-activity-free survival 85% vs. 59%; HR 0.13, 95% CI 0.03-0.59; P = 0.009). The ARR at 36 months was significantly lower in the aHSCT group (0.05 vs. 0.35, P = 0.02). A significant effect of aHSCT in promoting EDSS improvement compared with alemtuzumab was noted (P = 0.035). CONCLUSIONS: Alemtuzumab and aHSCT are effective treatment choices for aggressive multiple sclerosis. aHSCT seems to be superior to alemtuzumab in inducing complete disease control and in promoting short-term disability improvement.

Autologous haematopoietic stem cell transplantation with an intermediate intensity conditioning regimen in multiple sclerosis: the Italian multi-centre experience.

BACKGROUND: Over recent years numerous patients with severe forms of multiple sclerosis (MS) refractory to conventional therapies have been treated with intense immunosuppression followed by autologous haematopoietic stem cell transplantation (AHSCT). The clinical outcome and the toxicity of AHSCT can be diverse, depending on the various types of conditioning protocols and on the disease phase. OBJECTIVES: To report the Italian experience on all the consecutive patients with MS treated with AHSCT with an intermediate intensity conditioning regimen, named BEAM/ATG, in the period from 1996 to 2008. METHODS: Clinical and magnetic resonance imaging outcomes of 74 patients were collected after a median follow-up period of 48.3 (range = 0.8-126) months. RESULTS: Two patients (2.7%) died from transplant-related causes. After 5 years, 66% of patients remained stable or improved. Among patients with a follow-up longer than 1 year, eight out of 25 subjects with a relapsing-remitting course (31%) had a 6-12 months confirmed Expanded Disability Status Scale improvement > 1 point after AHSCT as compared with one out of 36 (3%) patients with a secondary progressive disease course (p = 0.009). Among the 18 cases with a follow-up longer than 7 years, eight (44%) remained stable or had a sustained improvement while 10 (56%), after an initial period of stabilization or improvement with median duration of 3.5 years, showed a slow disability progression. CONCLUSIONS: This study shows that AHSCT with a BEAM/ATG conditioning regimen has a sustained effect in suppressing disease progression in aggressive MS cases unresponsive to conventional therapies. It can also cause a sustained clinical improvement, especially if treated subjects are still in the relapsing-remitting phase of the disease.

KV7 channels regulate muscle tone and nonadrenergic noncholinergic relaxation of the rat gastric fundus.

Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13-25% or reduced by 11-21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.

Evaluation of Kisspeptin levels in prepubertal obese and overweight children: sexual dimorphism and modulation of antioxidant levels.

OBJECTIVE: Kisspeptin, neuropeptide involved in puberty beginning and regulation of pituitary-gonadal axis, has been shown to stimulate antioxidant defenses in murine models. Its levels are greater in females than males and also in obese prepubertal girls. Therefore, our aim was to evaluate sex-related differences in prepubertal obese patients and the relationships of Kisspeptin with metabolic/hormonal parameters. PATIENTS AND METHODS: We studied Kisspeptin concentrations in 54 children (22 males and 32 females, Tanner stage 1), 5-12 ys, classified according to Cole's criteria into 17 overweight and 37 obese; 25 normal-weight children, aged 6-12 years, were studied as controls. We evaluated metabolic (glucose and insulin levels after oral glucose load, total- LDL- HDL-cholesterol, triglycerides, uric acid) and hormonal (fT3, fT4, TSH, IGF-1, leptin) parameters. Moreover, total antioxidant capacity (TAC) was evaluated by spectrophotometric method, using the system H202-metmyoglobin-ABTS. Kisspeptin levels were measured by RIA. RESULTS: We did not find significant differences between obese and normal-weight children, but obese males presented significantly lower levels than females. Kisspeptin did not correlate with BMI, HOMA-IR, Insulin peak levels and TAC; a significant correlation was found between Kisspeptin and fT3 (r2=0.25; p=0.003) in the obese group; leptin levels, significantly greater in obese vs. overweight and control children, significantly correlated with TAC (r2=0.39; p=0.03). CONCLUSIONS: These data suggest that both hormones could modulate antioxidants, Kisspeptin indirectly via influence on thyroid hormones, and Leptin by a direct effect. This mechanism seems to be sex-related, not attributable to peripheral steroid levels. Further studies can clarify the complex interrelationship between central and peripheral Kisspeptin secretion and oxidative stress in children obesity.

Neurotransmitters of the non-adrenergic non-cholinergic relaxation of proximal stomach.

The proximal third of the stomach (fundus plus oral corpus) relaxes during swallowing so that it can hold large amounts of food with limited increases in intraluminal pressure. This mechanism has been called "receptive relaxation" and is mediated by a vago-vagal reflex. When the food bolus reaches the stomach, gastric relaxation is maintained by another reflex starting from mechanoreceptors in the gastric wall. This second mechanism has been named "adaptive relaxation" or "gastric accommodation" and involves both intramural and vagal reflex pathways, whose inhibitory neurons are always intramural. There was initially a great deal of controversy about the identity of the neurotransmitter/s released by inhibitory neurons, but at present nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are considered to be the most likely candidates. Several lines of evidence indicate that adenosine triphosphate (ATP) might be implicated too. It seems that these neurotransmitters are co-released from the inhibitory motor neurons and are responsible for the different features of the NANC relaxation induced by low- or high-frequency neuronal firing. NO (and perhaps ATP) would be responsible for the rapid beginning and the initial rapid development of the relaxation evoked by neuronal firing at low- or high-frequency and VIP for the long duration of the relaxation evoked by high-frequency neuronal activation. This review will deal mainly with the physiological characteristics and pharmacological features of the NANC relaxation of the proximal stomach and the evidences favoring or excluding a role as inhibitory neurotransmitters of ATP, NO and VIP in different species.

Expression of iNOS, CD163 and ARG-1 taken as M1 and M2 markers of microglial polarization in human glioblastoma and the surrounding normal parenchyma.

Microglia and macrophages appear to be the most common cells in the GBM microenvironment. In the present study we investigated the status of macrophages/microglia activation in surgical specimens from 41 patients diagnosed with grade IV GBM. For each patient we analyzed both the center of tumor and the parenchyma surrounding the tumor. The specimens were stained for: i) IBA1, a 17-kDa EF hand protein specifically expressed in microglia/macrophages ii) CD163, a cell surface antigen associated with M2 phenotype; iii) iNOS, taken as a functional marker of M1 phenotype, and iv) ARG-I, taken as a functional marker of M2 phenotype. Staining was scored in a double-blinded score on a scale from 0 to 5. Our results suggest that CD163 expression is higher within the tumor than in surrounding periphery in both male and female patients; while iNOS is higher within the tumor in males, no significant difference was found for ARG-1. In addition, analyzing the data in TGCA database, we found that CD163 expression was significantly and inversely correlated with mean survival times, with average survival times ranging from 448days in patients having low expression, to 319 in mid, and 353 in patients with high CD163 expressing tumors. In contrast, no significant association was found between survival time and ARG-1 or iNOS expression.

Predictors of failure after single faecal microbiota transplantation in patients with recurrent Clostridium difficile infection: results from a 3-year, single-centre cohort study.

OBJECTIVES: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (CDI). Although a single faecal infusion is usually sufficient to eradicate CDI, a considerable number of patients need multiple infusions to be cured. The aim of this study was to identify predictors of failure after single faecal infusion in patients with recurrent CDI. METHODS: We included patients with recurrent CDI prospectively treated with FMT by colonoscopy. By means of univariate and multivariate analysis, variables including female gender, age, number of CDI recurrences, severity of CDI, hospitalization, inadequate bowel preparation, unrelated donor, and use of frozen faeces, were assessed to predict failure after single faecal infusion. RESULTS: Sixty-four patients (39 women; mean age 74 years) were included. Of them, 44 (69%) were cured by a single faecal infusion, whereas 20 (31%) needed repeat infusions. Overall, FMT cured 62 of 64 (97%) patients. In the subgroup of patients with severe CDI, only eight of 26 (30%) were cured with a single infusion. At multivariate analysis, severe CDI (OR 24.66; 95% CI 4.44-242.08; p 0.001) and inadequate bowel preparation (OR 11.53; 95% CI 1.71-115.51; p 0.019) were found to be independent predictors of failure after single faecal infusion. CONCLUSIONS: Severe CDI and inadequate bowel preparation appear to be independent predictors of failure after single faecal infusion in patients treated with FMT by colonoscopy for recurrent CDI. Our results may help to optimize protocols and outcomes of FMT in patients with recurrent CDI.

Molecular cloning of the orphanin FQ receptor gene and differential tissue expression of splice variants in rat.

Pharmacological and receptor-ligand binding studies of the cloned orphanin FQ (OFQ) receptor suggest that multiple forms of this receptor may exist. To further characterize the OFQ receptor (OFQR), we attempted to isolate the gene encoding this receptor in rat. The OFQR gene exceeds 10 kb in length and contains six exons ranging from 34 to 524 bp that are interrupted by five introns. The ATG translation initiation codon is located in exon 2, and the open reading frame consists of 1283 bp. Primer extension analysis of the gene revealed two major transcription initiation sites: one in the 5' flanking region and the other in intron 1. The rat OFQR gene appeared to be alternatively spliced to yield multiple mRNAs. Four splice variants deleted for exon 1 were expressed only in brain. In contrast, five isoforms containing exon 1 were expressed in various tissues, such as brain, testes, and gastrointestinal tract. These data suggest that unique regions in intron 1 and in the 5' flanking region of the OFQR gene contribute to the regulation of its expression in different tissues.

Involvement of vasoactive intestinal polypeptide in nicotine-induced relaxation of the rat gastric fundus.

1. Nicotine-induced relaxation and release of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus. 2. Under non-cholinergic conditions (0.3 microM atropine), nicotine (3-300 microM) produced concentration-dependent relaxations of the 5-hydroxytryptamine (3 microM)-precontracted strips. Under non-adrenergic non-cholinergic (NANC) conditions (0.3 microM atropine + 1 microM phentolamine + 1 microM nadolol), relaxations induced by sub-maximal nicotine concentrations (10 and 30 microM) were significantly smaller, while that produced by the highest concentration used (300 microM) was similar to that seen under non-cholinergic conditions. 3. Re-exposure to the same nicotine concentration 1 h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used. 4. Under non-cholinergic conditions, the relaxant response to 30 microM nicotine was abolished by hexamethonium (100 microM) and significantly reduced by tetrodotoxin (TTX, 3 microM). The TTX-resistant component was not observed under NANC conditions. 5. NANC relaxation induced by 30 microM nicotine was significantly reduced by a specific anti-VIP serum (approximately 35% less than that seen with normal rabbit serum). 6. Nicotine (30-300 microM) caused significant, concentration-dependent increases in the outflow of VIP- and PHI-LI from the strips; these effects were also diminished with re-exposure. The increases in both types of immunoreactivity evoked by nicotine (300 microM) were abolished by hexamethonium (300 microM), TTX (3 microM) and a calcium-free medium. 7. These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.

Peptide histidine isoleucine-like immunoreactivity release from the rat gastric fundus.

1. Longitudinal muscle strips from the rat gastric fundus were subjected to in vitro electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions to study the release of peptide histidine isoleucine-like immunoreactivity (PHI-LI) and the correlation between PHI-LI release and NANC relaxation. 2. Different radioimmunoassay (RIA) systems employing C-terminal- and N-terminal-specific anti-PHI sera were used to determine the relative contributions of PHI and its C-terminally extended forms, peptide histidine glycine (PHI-Gly) and peptide histidine valine [PHV(1-42)], to the PHI-LI released by the rat gastric fundus. 3. In the presence of atropine (1 microM) and guanethidine (5 microM), EFS (120 mA, 1 ms, 0.25-32.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM) pre-contracted strips. 4. EFS at frequencies of 8-32 Hz evoked significant increases in PHI-LI outflow. The increases in PHI-LI outflow evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM) and by a calcium-free medium, indicating an active release process from intramural nerves. 5. The EFS-induced release of PHI-LI measured with the N-terminal-specific antiserum was significantly greater than that detected with the C-terminal-specific antisera. 6. Sephadex G-25 gel permeation chromatographic analysis was performed on the PHI-LI release in response to 32-Hz EFS. A C-terminal-specific antiserum revealed one peak co-eluting with the rat PHI standard. When PHI-LI was measured with the N-terminal-specific antiserum, two peaks were found that co-eluted with the rat PHV(1-42) and rat PHI-Gly/PHI standards, respectively. 7. The present data suggest that the extended forms of PHI are the primary components of the PHI-LI released by NANC inhibitory neurones in the rat gastric fundus and support a NANC inhibitory neurotransmitter role for PHI and its extended forms in this tissue.

Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus.

1. In the presence of atropine (1 microM) and guanethidine (5 microM), electrical field stimulation (EFS, 120 mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM)-precontracted longitudinal muscle strips from the rat gastric fundus. 2. L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adrenergic non-cholinergic (NANC) relaxation. 3. Basal NO synthase activity was reflected by the finding of prestimulation levels of L-citrulline of approximately 30 nM. These levels were unaffected by tetrodotoxin (3 microM) and NG-nitro-D-arginine methyl ester (D-NAME, 100 microM), slightly reduced by a calcium-free medium and halved by NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). 4. EFS evoked significant, frequency-dependent increases in bath levels of L-citrulline at all frequencies tested. The increases evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM), a calcium-free medium and L-NAME (100 microM) but not by D-NAME (100 microM). 5. L-NAME (0.1 microM-1.0 mM) produced significant reduction of 4-Hz EFS-induced L-citrulline production (100% inhibition at 10 microM), but had less marked effects on basal production (approximately 50% reduction at 100 microM) and 4-Hz EFS-induced NANC relaxation (approximately 50% reduction at 1 mM). 6. L-Arginine (1 mM), but not D-arginine (1 mM), increased basal L-citrulline levels and reversed the inhibitory effect of L-NAME (10 microM). 7. These findings represent clear biochemical evidence of both basal and EFS-stimulated NO synthase activity in the rat gastric fundus.

Effects of vasoactive intestinal polypeptide on antigen-induced bronchoconstriction and thromboxane release in guinea-pig lung.

1. Exogenous vasoactive intestinal polypeptide (VIP) infused into the pulmonary artery of isolated and ventilated lungs of guinea-pigs decreased, in a dose-dependent fashion (1.0-10.0 nmol), airway resistance and thromboxane B2 (TXB2, the stable hydrolysis product of TXA2) release in the perfusion medium. Prostacyclin (PGI2) synthesis, as reflected by the release of its stable hydrolysis product 6-oxo-PGF1 alpha, was unaffected. Pretreatment with the 5-lipoxygenase inhibitor BWA4c (3.5 x 10(-5) M) did not modify the bronchodilatory effect of VIP or its inhibitory action on TXB2 release. 2. Basal release of immunoreactive VIP from perfused lungs decreased from an initial value of 0.96 +/- 0.10 ng min-1 (mean +/- s.e.mean) in the first 2 min to an average of 0.58 +/- 0.10 ng min-1 in the following 15-20 min. 3. Antigen challenge with ovalbumin (0.1%) in sensitized lungs caused an anaphylactic reaction in 45% of tested lungs, concomitant with a 5 fold increase in both VIP and TXB2 release. Tetrodotoxin pretreatment (10(-6) M) reduced basal VIP release by > 80% and abolished the VIP increase observed during anaphylaxis, without modifying TXB2 release or the bronchoconstrictor response. 4. Indomethacin (10(-6) M) inhibited TXB2 synthesis and release by > 90%, delayed the bronchoconstrictor response and blunted the increased VIP release during lung anaphylaxis, without influencing basal VIP release. 5. The 5-lipoxygenase inhibitor BWA4c (3.5 x 10(-5) M) blunted the increase of TXB2 and VIP release from guinea-pig lung and attenuated the bronchoconstrictor response following ovalbumin challenge. 6. The administration of exogenous VIP as a continuous infusion (10-8 M) attenuated the bronchoconstriction and the release of cyclo-oxygenase metabolites following antigen challenge.7. Acetylcholine (10-6-l0-5 M) infused into the pulmonary artery induced a dose-dependent bronchoconstriction not associated with enhanced VIP or TXB2 release.8. The TXA2 mimetic U-46619 (0.1-1.0 nmol) caused dose-dependent increases in airway resistance,concomitant with an up to 10 fold increase in VIP release. VIP inhibited arachidonate-induced in vitro aggregation of washed rabbit platelets in a dose-dependent manner over a dose range 10-8 10-6 M.Despite the antiaggregatory effect of VIP, TXB2 and PGE2 synthesis was reduced only to a minor extent,and there was no redirection of arachidonate metabolism from TXA2 to PGE2, indicating that VIP does not act as a TX synthase inhibitor in vitro.9. We conclude that VIP may play a role in regulating bronchial smooth muscle reactivity in lung anaphylaxis by inhibiting the synthesis and release of TXA2, a potent vasoactive and bronchoconstrictor agent. TXA2, on the other hand, strongly enhances neuronal VIP release.

Release of vasoactive intestinal polypeptide from the rat gastric fundus.

1. Auxotonic responses and release of vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) induced by electrical field stimulation (EFS) were studied in longitudinal muscle strips from the gastric fundus of reserpinized rats suspended between parallel platinum electrodes in Krebs solution containing atropine (1 microM), 5-hydroxytryptamine (3 microM) and bovine serum albumin (50 mg l-1). 2. EFS (supramaximal voltage, 1 ms, 0.25-32.0 Hz, trains of 2 min) induced frequency-dependent relaxations. 3. EFS at frequencies greater than or equal to 8 Hz also produced significant increases in VIP-LI release. 4. VIP-LI release induced by EFS at 16 Hz no longer occurred in the presence of tetrodotoxin (1 microM) or a Ca(2+)-free medium. 5. Detection of VIP-LI upon activation of inhibitory non-adrenergic, non-cholinergic neurones indicates that VIP meets the 'detectable release' criterion for an inhibitory neurotransmitter in the rat gastric fundus.

Voltage-operated Ca(2+) channels involved in K(+)-evoked release of vasoactive intestinal polypeptide from the rat hypothalamus.

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.

Time dependence of endothelium-mediated vasodilation by intermittent antegrade warm blood cardioplegia.

BACKGROUND: The technique of intermittent antegrade warm blood cardioplegia (IAWBC) exposes the heart to brief periods of normothermic ischemia. This may impair endothelial function in coronary arteries. METHODS: Three cardioplegic technique were tested in porcine hearts arrested for 32 to 36 minutes and reperfused for 30 minutes: IAWBC, antegrade cold blood cardioplegia (ACBC), and antegrade cold crystalloid cardioplegia (ACCC). In the hearts arrested with IAWBC, three different intervals of ischemia were used: three 10-minute intervals (IAWBC1), two 15-minute intervals (IAWBC2), and one 30-minute interval (IAWBC3). Rings from the coronary arteries were used to evaluate in vitro the contractile responses to U46619 and the relaxant responses to bradykinin, A23187, and sodium nitroprusside. RESULTS: All six groups (treatment groups and control group) displayed similar responses to U46619 (30 nmol/L) and nitroprusside. In the IAWBC1, IAWBC2, AND ACBC groups, endothelium-dependent relaxations to bradykinin and A23187 were preserved compared with controls, whereas those of the ACCC and IAWBC3 groups were significantly impaired (bradykinin: control, 8.72 +/- 0.07; IAWBC1, 8.73 +/- 0.03; IAWBC2, 8.65 +/- 0.05; IAWBC3, 8.30 +/- 0.07 [p < 0.05]; ACBC, 8.50 +/- 0.03; ACCC, 8.25 +/- 0.09 [p < 0.05]; A23187: control, 7.07 +/- 0.08; IAWBC1, 7.07 +/- 0.06; IAWBC2, 7.04 +/- 0.03; IAWBC3, 6.64 +/- 0.01 [p < 0.05]; ACBC, 6.80 +/- 0.05; ACCC, 6.60 +/- 0.08 [p < 0.05]; nitroprusside: control, 6.19 +/- 0.1; IAWBC1, 6.19 +/- 0.07; IAWBC2, 6.03 +/- 0.03; IAWBC3, 6.08 +/- 0.05; ACBC, 6.04 +/- 0.2; ACCC, 6.05 +/- 0.03; all values are expressed as the negative logarithm of the concentration producing 50% of the maximal response). CONCLUSIONS: Myocardial preservation with IAWBC with ischemic intervals of 15 minutes or shorter does not alter the endothelium-dependent relaxation to bradykinin or A23187 in porcine coronary arteries, but these responses are significantly impaired by ACCC and IAWBC with an ischemic interval of 30 minutes.

Tachykinin NK2 receptor antagonists decrease eicosanoid release in lung anaphylaxis.

Two tachykinin NK2 receptor antagonists, MEN 10.627 c(Met-Asp-Trp-Phe-Dap-Leu) and MEN 10.376 [(Tyr5,Trp6,8,9,Lys10]neurokinin A-(4-10), were used to investigate the role of tachykinins in in vitro guinea-pig lung anaphylaxis. Both antagonists dose-dependently decreased bronchoconstriction and the release of thromboxane and prostaglandin E2 induced by antigen challenge in perfused sensitized lungs, but neither had any effect on the basal release of either eicosanoid. The findings indicated that tachykinins released by sensory nerve fibers may contribute to anaphylactic reactions by increasing arachidonic acid metabolite release.

Anaphylaxis increases 8-iso-prostaglandin F2alpha release from guinea-pig lung in vitro.

8-Iso-prostaglandin F2alpha, release from isolated and perfused guinea-pig lung was measured by radioimmunoassay. 8-Iso-prostaglandin F2alpha release was detectable under basal conditions and increased 10-fold during antigen-induced bronchoconstriction, concomitant with the increase of thromboxane B2 and prostaglandin E2. The anti-8-iso-prostaglandin F2alpha serum used in the radioimmunoassay seems to be quite specific for this compound. Pretreatment of lungs with indomethacin (a non-selective inhibitor of cyclooxygenase) reduced 8-iso-prostaglandin F2alpha release under basal conditions and completely abolished the increase observed during lung anaphylaxis. Pretreatment of lungs with NS 398 (N-[2-cyclohexyl]-4-nitrophenyl methanesulphonamide), a selective inhibitor of cyclooxygenase-2, did not change basal or antigen-induced 8-iso-prostaglandin F2alpha release at all. We conclude that under basal conditions guinea-pig lung perfusates contain low levels of 8-iso-prostaglandin F2alpha-like immunoreactivity, which increase 10-fold during antigen-induced bronchoconstriction. This isoprostane seems to be derived from the cyclooxygenation of arachidonic acid via the constitutive form of cyclooxygenase.

Evidence that interleukin-1 beta and tumor necrosis factor inhibit gastric fundus motility via the 5-lipoxygenase pathway.

In this study, we compared the effects of interleukin-1 beta and tumor necrosis factor (TNF) on in vitro rat gastric fundus motility. Interleukin-1 beta produced rapid, concentration-dependent relaxation of rat gastric fundus strips, similar to that seen with TNF, with a maximal effect at 30 U/ml and an estimated EC50 at 0.9 U/ml. The relaxant effects of interleukin-1 beta and TNF were not influenced by the inhibition of cyclooxygenase or nitric oxide-synthase activities. Interleukin-1 beta- and TNF-induced gastric relaxations were concentration dependently inhibited by BW 755c, which inhibits both cyclooxygenase and lipoxygenase, BW A4, which selectively inhibits the 5-lipoxygenase pathway, and SC 41930, a selective leukotriene B4 receptor antagonist, providing pharmacological evidence that leukotriene B4 is involved in the relaxant effects of both cytokines. The interleukin-1 beta- and TNF-induced activation of 5-lipoxygenase pathway did not appear to be triggered by phospholipase A2. An alternative pathway could involve the following steps: (i) activation of phospholipase C and the formation of diacylglycerol; (ii) diacylglycerol-induced activation of protein kinase C; (iii) formation of free arachidonic acid from diacylglycerol by diacylglycerol-lipase. This mechanism is suggested by the finding that leukotriene B4 is able to mimic cytokine-induced strip relaxation only in the presence of phorbol 12-myristate 13-acetate, which selectively activates protein kinase C.

In vitro testing for lung toxicity.

We characterized the pattern of arachidonic acid metabolites released from sensitized isolated guinea pig lungs undergoing anaphylactic reactions after ovalbumin challenge. TXB(2) (the stable hydrolysis product of (TXA(2)) is the most abundant product: it was released at an average of 13.7 +/- 7.0 ng/min (+/-SD, n = 33) during the anaphylactic reaction (i.e. a five- to six-fold increase in comparison with the basal release). Its level correlates with both the bronchoconstrictor response and LTC(4) and LTB(4) release. In non-sensitized lungs the release of basal TXB(2) increased about five-fold during a 10-min exposure to a formaldehyde aerosol at the dose of 10 ppm; this increase was concomitant with an 87.5 +/- 10.0% (+/-SD) reduction of the tracing recording the pressure-volume variations in the lung. No change in the release of lipoxygenase products was observed after exposure to formaldehyde aerosol. Pre-treatment with N-acetylcysteine (10(-3)-10(-4)m) reduced both formaldehyde-induced bronchoconstriction and the increase of TXB(2) release. An acid-water aerosol, resembling the composition of acid rain, strongly increased TXB(2) release in the pH range 4.5 to 2.5, without affecting the lipoxygenase pathway of arachidonic acid metabolism. These data suggest that exposure to toxicants that irritate the respiratory tract selectively enhances TXB(2) release, while an antigen challenge stimulates both the cyclooxygenase and lipoxygenase pathways of lung arachidonate metabolism. Thus, the different pattern of arachidonic acid cascade activation may predict a direct toxic effect or an allergic reaction when the xenobiotic is injected into the pulmonary artery or administered by way of inhalation as an aerosol. More recently vasoactive intestinal polypeptide (VIP) has been proposed as a modulator of lung inflammation and airway constriction. In sensitized lungs the antigen challenge induced a five-fold increase in VIP release, coinciding with the increase of arachidonate metabolites. Pharmacological evidence shows that TXA(2) enhances VIP release, while VIP suppresses TXA(2) formation. This suggests a possible role of TXA(2)/VIP interaction in regulating bronchial smooth muscle reactivity in condition of enhanced arachidonate metabolism. However, the importance of this interaction in evaluating the toxic response is still to be established.