Atrial natriuretic peptide reduces inflammation and enhances apoptosis in rat acute pancreatitis.

AIM: We previously reported that atrial natriuretic peptide (ANP) reduces serum amylase and intrapancreatic trypsinogen activation in the onset of acute pancreatitis whereas secretin increases them. In the present work, we sought to establish the effect of ANP and secretin on the inflammatory response and cell death in experimental acute pancreatitis. METHODS: The expression and activity of key inflammatory mediators and apoptosis were evaluated in the presence or absence of the atrial peptide, secretin or both in cerulein-induced acute pancreatitis in rats. Also, ultrastructural changes in pancreatic acinar cells were assessed by transmission electron microscopy. RESULTS: ANP significantly reduced NF-κB activation and TNF-α intrapancreatic levels. Furthermore, it decreased inducible nitric oxide synthase and cyclooxygenase 2 expression and activity while it diminished myeloperoxidase activity. ANP also stimulated apoptosis as shown by caspase-3 expression and activation as well as TUNEL assay. These findings correlated well with the ultrastructural changes observed in the exocrine pancreas. Although secretin reduced various inflammatory markers, it also diminished caspase-3 activation and the overall response was the aggravation of the disease as reflected by the ultrastructural alterations of pancreatic acinar cells. In the presence of ANP, various effects evoked by secretin were antagonized. CONCLUSION: Present findings show that ANP significantly attenuated the severity of acute pancreatitis in the rat by inducing apoptosis and reducing the inflammatory response and further suggest that ANP may have eventual therapeutic implications in the disease and/or in medical interventions at risk of its developing like endoscopic retrograde cholangiopancreatography.

Endothelin-mediated calcium responses in supraoptic nucleus astrocytes influence magnocellular neurosecretory firing activity.

In addition to their peripheral vasoactive effects, accumulating evidence supports an important role for endothelins (ETs) in the regulation of the hypothalamic magnocellular neurosecretory system, which produces and releases the neurohormones vasopressin (VP) and oxytocin (OT). Still, the precise cellular substrates, loci and mechanisms underlying the actions of ETs on the magnocellular system are poorly understood. In the present study, we combined patch-clamp electrophysiology, confocal Ca(2+) imaging and immunohistochemistry to study the actions of ETs on supraoptic nucleus (SON) magnocellular neurosecretory neurones and astrocytes. Our studies show that ET-1 evoked rises in [Ca(2+) ](i) levels in SON astrocytes (but not neurones), an effect largely mediated by the activation of ET(B) receptors and mobilisation of thapsigargin-sensitive Ca(2+) stores. The presence of ET(B) receptors in SON astrocytes was also verified immunohistochemically. ET(B) receptor activation either increased (75%) or decreased (25%) SON firing activity, both in VP and putative OT neurones, and these effects were prevented when slices were preincubated in glutamate receptor blockers or nitric oxide synthase blockers, respectively. Moreover, ET(B) -mediated effects in SON neurones were also prevented by a gliotoxin compound, and when changes in [Ca(2+) ](i) were prevented with bath-applied BAPTA-AM or thapsigargin. Conversely, intracellular Ca(2+) chelation in the recorded SON neurones failed to block ET(B) -mediated effects. In summary, our results indicate that ET(B) receptor activation in SON astrocytes induces the mobilisation of [Ca(2+) ](i) , likely resulting in the activation of glutamate and nitric oxide signalling pathways, evoking in turn excitatory and inhibitory SON neuronal responses, respectively. Taken together, our study supports an important role for astrocytes in mediating the actions of ETs on the magnocellular neurosecretory system.

Human herpes virus 6 in archival cardiac tissues from children with idiopathic dilated cardiomyopathy or congenital heart disease.

OBJECTIVE: To explore the possible role of human herpes virus 6 (HHV-6) in cardiac disorders in childhood in a retrospective study on archival specimens of explanted hearts. METHODS: 16 children (median age at transplantation 11.0 years) with idiopathic dilated cardiomyopathy (DCM) and 19 children (median age at transplantation 1.0 year) with congenital heart disease (CHD), previously found to be negative for other cardiotropic viruses such as enteroviruses, adenovirus, parvovirus B19, cytomegalovirus and Epstein-Barr virus, were tested for HHV-6 by quantitative real-time PCR and by genotyping. In addition, HHV-7/8 infection was investigated by qualitative PCR. RESULTS: HHV-6 B variant was detected in 11 of 35 samples (31.4%) with a mean viral load of 3.1 x 102 copies/microg of DNA. When assessed by heart disorder, the prevalence was different in the two groups (43.7% in DCM and 21% in CHD) while the mean viral loads were similar. In a logistic multivariate analysis HHV-6 was independently associated with DCM, taking CHD as reference and adjusting for age (best estimate: OR = 6.94; 95% CI 1.00 to 49.85; p = 0.05). CONCLUSIONS: Although the clinical significance of the results is unknown, HHV-6 B genome is frequently detected in explanted hearts from children with DCM and to a lesser extent with CHD, thus adding evidence for HHV-6 cardiac involvement.

A novel mutation of the fibrillin-1 gene in a newborn with severe Marfan syndrome.

Marfan syndrome in the neonatal age represents a severe early and commonly lethal manifestation of Marfan syndrome, which is caused by mutations in the gene encoding fibrillin-1 (FBN1). Here, we report a newborn with severe Marfan syndrome and a novel mutation involving cysteine substitution within one of the epidermal growth factor-like domains of FBN1.

Protein free diet feeding: effects on sympathetic activity and salivary evoked secretion in the submandibular gland of the rat.

Protein restriction impairs the salivary flow rate and composition in human and rats. The aim of the present work was to establish the effect of low protein (casein 5%) and protein free (casein 0%) isocaloric diets on sympathetic activity and salivary evoked secretion in the submandibular gland (SMG) of the rat. After 21 days, rats fed casein 0% presented: (a) a significant shift to the left of the dose-response curves (DRC) to the autonomic agonists-norepinephrine (NE), methoxamine, isoproterenol (ISO) and methacholine; (b) increased food consumption (p<0.001); (c) decreased body (p<0.001) and SMG (p<0.001) weights maintaining SMG/body (w/w) relation; (d) enhanced submandibular alpha1-adrenoceptor number without changes in the apparent dissociation constant (Kd); (e) increased submandibular NE content (p<0.05) and phosphoinositoside hydrolysis (p<0.001); (f) decreased submandibular tyrosine hydroxylase activity (TH) (p<0.01). Casein 5% feeding increased food consumption (p<0.01) and reduced body weight (p<0.05). This protein restriction increased metacholine-evoked salivation, but it altered neither submandibular sympathetic activity nor sympathetic-induced salivary secretion as compared to the Control group (C) fed a similar diet containing 25.5% protein. Present results suggest that in the adult rat, a protein free diet during 21 days lowers SMG sympathetic and cholinergic activity leading to supersensitivity as revealed by up-regulation of alpha1-adrenergic receptor number and increased autonomic-evoked salivation.

Possible association of the human KCNE1 (minK) gene and QT interval in healthy subjects: evidence from association and linkage analyses in Israeli families.

QT interval prolongation is associated with increased risk of sudden and non-sudden cardiac death. Potassium channel gene variants are associated with inherited long QT syndromes. Using linkage and association analyses, we investigated whether variants in the potassium channel subunit KCNE1 are associated with QTc intervals in an unselected population sample of 80 kindreds living in kibbutz settlements in Israel. Variance-component linkage analysis revealed weak evidence of linkage of KCNE1 polymorphisms with QTc intervals. Family-based association analysis showed a significant association between the G38S polymorphism and QTc interval. Further quantitative trait association analysis demonstrated a significant residual heritability component (h(2)= 0.33), and that the effect of the G38S variant allele is modified by gender. Estimated maximum likelihood parameters from these models indicated that male gender, age, hypertension, diabetes, hypercholesterolemia, fibrinogen and BMI were positively associated with QTc interval; level of education and cigarette smoking showed an inverse association. Both erythrocyte membrane n-6 and n-3 fatty acids showed a significant inverse association with QTc interval. While more than 15.8% of QTc variability was contributed by covariates, another 4.7% was explained by dietary factors, the G38S polymorphism explained 2.2%, and approximately 36% was explained by polygenes. An in silico analysis showed also that the novel V80 SNP, another KCNE1 synonymous variant, abolishes the recognition for a splicing enhancer, which may lead to an increased effect of the G38S mutation. These results demonstrate that, in addition to polygenic background, dietary factors and other covariables, the KCNE1 G38S variant is involved in determining QTc levels in this population-based sample of families.

Altered dystrophin expression in the right atrium of a patient after Fontan procedure with atrial flutter.

Underlying mechanisms in the development of atrial flutter or intra-atrial re-entry tachycardia in patients with structural cardiac abnormalities remain poorly defined. The right atrial myocardium from two patients with congenital heart disease was evaluated, of whom one presented with severe right atrial dilation and arrhythmia and the other with a normal right atrium, to assess whether increased right atrial pressure and volume overload give rise to sarcolemmal alteration. N-terminus dystrophin staining in the atrium from the patient who had undergone a Fontan procedure showed a normally distributed but significantly reduced staining signal compared with the second patient. This is the first report that patients with severe right atrial dilation and atrial flutter have marked reduction in atrial dystrophin expression.

Salivary glands and noradrenergic transmission in diabetic rats.

1 Type 2 diabetes is associated with diverse oral pathologies in which salivary flow reduction is one of the causes of these oral abnormalities. Scarce literature exists regarding noradrenergic transmission and adrenergic-induced salivary flow in submaxillary and parotid glands of type 2 diabetic rats. 2 We studied noradrenergic transmission as well as the secretory response to alpha1- and beta-adrenoceptor stimulation in the parotid and submaxillary glands of type 2 diabetic rats. 3 Diabetic rats exhibited diminished neuronal uptake, release and endogenous content of noradrenaline (NE) in both salivary glands. Further, NE synthesis was also diminished accompanied by decreased tyrosine hydroxylase activity. Salivary flow responses to alpha1-(methoxamine) and beta-(isoprenaline) adrenoceptor stimulation were reduced in the submaxillary as well as the parotid glands of diabetic rats. 4 Our results suggest that the reduction of noradrenergic transmission in the salivary glands of type 2 diabetic rats is in part responsible for the diminished salivary flow evoked by alpha1- and beta-adrenergic stimulation. Reduced noradrenergic activity may contribute to the pathophysiology of oral abnormalities in diabetic patients.

Cyclosporin A: effects on the secretory process and noradrenergic activity in the submandibular gland of the rat.

OBJECTIVES: The aim of the present work was to study the effect of long-term cyclosporine (CSA) administration on norepinephrine (NE) metabolism and adrenergic-evoked secretion in the rat submandibular gland (SMG). METHODS: Dose-response curves to adrenergic agonists (methoxamine, isoproterenol, NE) were performed in control and CSA (10 and 30 mg/kg every 2 days for 1 month)-treated rats after SMG duct cannulation. In SMG tissue neuronal NE uptake, release, synthesis and endogenous content were determined. In addition phosphoinositide intracellular signaling was also investigated. RESULTS: CSA administration caused an increase in salivary secretion evoked by methoxamine (alpha-adrenergic agonist) and NE but failed to modify salivation evoked by beta-adrenergic stimulation (isoproterenol). Long-term CSA administration decreased NE release and synthesis whereas it enhanced the amine uptake and phosphoinositide hydrolysis in the SMG. CONCLUSIONS: The administration of CSA for 30 days induced salivary gland sensitization likely mediated by diminished adrenergic input. Present results suggest that the decreased sympathetic activity evoked by long-term CSA administration in the rat SMG may lead to sensitization of the gland supported by increased phosphoinositide hydrolysis and enhanced adrenergic-evoked salivation.

Centrally applied atrial natriuretic factor diminishes bile secretion in the rat.

Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor (ANF) reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ng/microl ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway.

Molecular biology and the prolonged QT syndromes.

The prolonged QT syndromes are characterized by prolongation of the QT interval corrected for heart rate (QTc) on the surface electrocardiogram associated with T-wave abnormalities, relative bradycardia, and ventricular tachyarrhythmias, including polymorphic ventricular tachycardia and torsades de pointes. These patients tend to present with episodes of syncope, seizures, or sudden death typically triggered by exercise, emotion, noise, or, in some cases, sleep. These disorders of cardiac repolarization are commonly inherited, with the autosomal dominant form, Romano-Ward syndrome, most common. A rare autosomal recessive form associated with sensorineural deafness, Jervell and Lange-Nielsen syndrome, in which the cardiac disorder is autosomal dominant and deafness is a recessive trait, also occurs. The underlying genetic causes of these forms of prolonged QT interval syndromes are heterogeneous, with at least seven genes responsible for the clinical syndromes. All of the five genes identified to date encode ion channel proteins, suggesting this to be an ion channelopathy. In this review, the genetic basis of the prolonged QT interval syndromes will be discussed, genotype-phenotype correlations identified, and the approaches to genetic testing and treatments will be outlined.

AT-1 receptor and phospholipase C are involved in angiotensin III modulation of hypothalamic noradrenergic transmission.

1. We previously reported that angiotensin III modulates noradrenergic neurotransmission in the hypothalamus of the rat. In the present work we studied the effects of angiotensin III on norepinephrine release and tyrosine hydroxylase activity. We also investigated the receptors and intracellular pathways involved in angiotensin III modulation of noradrenergic transmission. 2. In rat hypothalamic tissue labeled with [3H]norepinephrine 1, 10, and 100 nM and 1 microM losartan (AT1 receptor antagonist) had no effect on basal neuronal norepinephrine release, whereas 10 and 100 nM and 1 microM losartan partially diminished norepinephrine secretion evoked by 25 mM KCl. The AT2 receptor antagonist PD 123319 showed no effect either on basal or evoked norepinephrine release. The increase in both basal and evoked norepinephrine output induced by 1 microM angiotensin III was blocked by 1 microM losartan, but not by 1 microM PD 123319. 3. The phospholipase C inhibitor 5 microM neomicin inhibited the increase in basal and evoked norepinephrine release produced by 1 microM angiotensin III. 4. Tyrosine hydroxylase activity was increased by 1 microM angiotensin III and this effect was blocked by 1 microM LST and 5 microM neomicin, but not by PD 123319. On the other hand, 1 microM angiotensin III enhanced phosphatidyl inositol hydrolysis that was blocked by 1 microM losartan and 5 microM neomicin. PD 123319 (1 microM) did not affect ANG III-induced phosphatidyl inositol hydrolysis enhancement. 5. Our results confirm that angiotensin III acts as a modulator of noradrenergic transmission at the hypothalamic level through the AT1-phospholipase C pathway. This enhancement of hypothalamic noradrenergic activity suggests that angiotensin III may act as a central modulator of several biological processes regulated at this level by catecholamines, such as cardiovascular, endocrine, and autonomic functions as well as water and saline homeostasis.

B-Type and C-type natriuretic peptides modify norepinephrine uptake in discrete encephalic nuclei of the rat.

1. We previously demonstrated that atrial natriuretic factor and B- and C-type natriuretic peptides (ANF, BNP, and CNP, respectively) modified catecholamine metabolism by increasing the neuronal uptake and decreasing the neuronal release of norepinephrine in the rat hypothalamus. The aim of the present work was to study the effects of natriuretic peptides BNP and CNP on norepinephrine uptake as an index of the amine metabolism in discrete areas and nuclei of the central nervous system (CNS) of the rat. 2. Experiments were carried out in vitro using the punchout technique in diverse areas and nuclei of rat CNS. Results showed that 100 nM BNP and 1 nM CNP increased norepinephrine (NE) uptake in all brain areas and nuclei studied. 3. Present results permit us to conclude that BNP and CNP regulate NE metabolism independently of the encephalic area or nucleus involved. In fact, NE uptake increased in nuclei related to the regulation of cardiovascular activity as well as nuclei associated with endocrine metabolism and hydrosaline homeostasis. These observations suggest that BNP and CNP may be involved in the regulation of these physiological processes in an indirect manner through modifications of noradrenergic neurotransmission. Present findings provide further support to the hypothesis that CNP would be the main natriuretic peptide in brain. Furthermore, previous as well as present results support the role of the natriureic peptides as neuromodulators of noradrenergic transmission at the presynaptic level.

Angiotensin-(1-7) does not affect norepinephrine neuronal uptake or catabolism in rat hypothalamus and atria.

1. Since we previously reported that angiotensin-(1-7) [Ang-(1-7)] increases or inhibits norepinephrine (NE) release in rat atria or hypothalamus, respectively, the present work was undertaken to investigate the effect of the heptapeptide on NE neuronal uptake and metabolism in atria and hypothalamus isolated from rats. 2. Ang II (1-10 microM) caused a decrease in neuronal NE uptake in both atria and hypothalami isolated from rats. On the contrary, tissues incubated with [3H]NE in the presence of 0.1-10 microM Ang-(1-7) showed no modification in [3H]NE content with respect to the control group, suggesting that the heptapeptide did not modify [3H]NE neuronal uptake. 3. To study the effect of the heptapeptide on NE catabolism, monoamine-oxidase (MAO) and catechol-O-methyltransferase (COMT) activities were determined. Pretreatment of the tissue with Ang-(1-7) (0.1-1.0 microM) showed a tendency to diminish MAO activity in rat atria, while no significant changes were observed in hypothalamic MAO activity. Moreover, the heptapeptide (0.1-1.0 microM) did not affect central COMT activity with respect to the control group. 4. Present results allow us to conclude that Ang-(1-7) interacts with noradrenergic neurotransmission by increasing or inhibiting NE release at the peripheral and central levels, respectively, without affecting either the neurotransmitter neuronal uptake or catabolism.

Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), whereas three other genes (actin, lamin A/C, and desmin) cause autosomal dominant DCM; seven other loci for autosomal dominant DCM have been mapped but the genes have not been identified. Hypothesizing that DCM is a disease of the cytoskeleton and sarcolemma, we have focused on candidate genes whose products are found in these structures. Here we report the screening of the human delta-sarcoglycan gene, a member of the dystrophin-associated protein complex, by single-stranded DNA conformation polymorphism analysis and by DNA sequencing in patients with DCM. Mutations affecting the secondary structure were identified in one family and two sporadic cases, whereas immunofluorescence analysis of myocardium from one of these patients demonstrated significant reduction in delta-sarcoglycan staining. No skeletal muscle disease occurred in any of these patients. These data suggest that delta-sarcoglycan is a disease-causing gene responsible for familial and idiopathic DCM and lend support to our "final common pathway" hypothesis that DCM is a cytoskeletalopathy.

Central natriuretic peptides regulation of peripheral atrial natriuretic factor release.

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP) receptors have been described in encephalic areas and nuclei related to the regulation of cardiovascular as well as sodium and water homeostasis. Stimulation of the anterior ventral third ventricular region of the brain modifies plasma ANF concentration, suggesting the participation of the central nervous system in the regulation of circulating ANF. The aim of this work was to study the effect of centrally applied ANF or CNP on plasma ANF. Normal and blood volume expanded rats (0.8 ml isotonic saline/100 g body weight) were intra cerebralventricularly injected with 1, 10 or 100 ng/microl/min ANF. Blood volume expanded animals were also centrally injected with the same doses of CNP. Blood samples were collected at 5 and 15 min. after intracerebralventricular administration of either ANF or CNP. Centrally applied ANF did not affect circulating ANF in normal blood volume rats. In blood volume expanded animals both ANF (1, 10 or 100 ng/microl/min) and CNP (1 ng/microl/min) decreased plasma ANF concentration after 15 min. Moreover, CNP (10 and 100 ng/microl/min) lowered circulating ANF levels not only at 15 min but also at 5 min. Neither ANF nor CNP elicited any change in mean arterial pressure and heart rate in normal and blood volume expanded rats. These results suggest the existence of a central regulation exerted by natriuretic peptides on circulating ANF levels. Furthermore, this is the first study reporting an effect on plasma ANF induced by centrally applied CNP.

Angiotensin-(1-7) reduces norepinephrine release through a nitric oxide mechanism in rat hypothalamus.

Angiotensin (Ang)-(1-7) elicits a facilitatory presynaptic effect on peripheral noradrenergic neurotransmission, and because biological responses to the heptapeptide on occasion are tissue specific, the present investigation was undertaken to study its action on noradrenergic neurotransmission at the central level. In rat hypothalamus labeled with [(3)H]-norepinephrine, 100 to 600 nmol/L Ang-(1-7) diminished norepinephrine released by 25 mmol/L KCl. This effect was blocked by the selective angiotensin type 2 receptor antagonist PD 123319 (1 micromol/L) and by the specific Ang-(1-7) receptor antagonist ([D-Ala(7)]Ang-(1-7) (1 micromol/L) but not by losartan (10 nmol/L to 1 micromol/L), a selective angiotensin type 1 receptor antagonist. The inhibitory effect on noradrenergic neurotransmission caused by Ang-(1-7) was prevented by 10 micromol/L N(omega)-nitro-L-arginine methylester, an inhibitor of nitric oxide synthase activity, and was restored by 100 micromol/L L-arginine, precursor of nitric oxide synthesis. Methylene blue (10 micromol/L), an inhibitor of guanylate cyclase considered as the target of nitric oxide action, as well as Hoe 140 (10 micromol/L), a bradykinin B(2)-receptor antagonist, prevented the inhibitory effect of the heptapeptide on neuronal norepinephrine release, whereas no modification was observed in the presence of 0.1 to 10 micromol/L indomethacin, a cyclooxygenase inhibitor. Our results indicate that Ang-(1-7) has a tissue-specific neuromodulatory effect on noradrenergic neurotransmission, being inhibitory at the central nervous system by a nitric oxide-dependent mechanism that involves angiotensin type 2 receptors and local bradykinin production.

Molecular biology of arrhythmic syndromes.

In this review, the up-to-date understanding of the molecular basis of primary ventricular arrhythmias will be outlined. Two disorders have recently been well described at the molecular level, the long QT syndromes and Brugada syndrome, and in this paper we review the current scientific knowledge of each disease.