Localization of pro-opiomelanocortin mRNA transcripts and peptide immunoreactivity in rat heart.

OBJECTIVE: alpha-Melanocyte-stimulating hormone (alpha-MSH), beta-endorphin and other pro-opiomelanocortin-(POMC) derived peptides have been detected in the heart, but it is uncertain whether they are synthesized by cardiomyocytes or by cardiac nerves innervating the heart. The objective of this study was to determine whether POMC peptides are synthesized by cardiomyocytes. METHODS: Pro-opiomelanocortin peptides were localized in rat heart by immunohistochemistry using antisera against alpha-MSH, beta-endorphin and alpha N-acetyl-beta-endorphin, the predominant POMC peptides found in heart. Pro-opiomelanocortin mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) using primers that discriminate between full-length POMC mRNA and a 5' truncated POMC transcript that is presumed to be non-functional. RESULTS: alpha-Melanocyte-stimulating hormone, beta-endorphin and alpha N-acetyl-beta-endorphin immunoreactivities were localized in atrial myocytes, particularly in the atrial appendages, but not to a significant extent in ventricular myocytes. Cardiac nerves were not immunostained. Atrial natriuretic peptide (ANP) immunoreactivity was similarly distributed in the adult heart. In neonatal heart, POMC-peptide and ANP immunoreactivities were present in both atrial and ventricular myocytes. RT-PCR amplification showed that full-length POMC mRNA transcripts were present in both atrial and ventricular tissue and provide evidence that 5' truncated POMC mRNA is expressed in heart. CONCLUSIONS: These results support the hypothesis that cardiomyocytes synthesize POMC peptides.

Differential effects of high salt intake on neuropeptide Y and adrenergic markers in hearts of Dahl rats.

Adrenergic markers and neuropeptide Y (NPY) were examined in Dahl NaCl-sensitive and -resistant outbred male rats, fed either 0.35% or 8% NaCl diets for 8 weeks. The high salt diet caused left ventricular hypertrophy in sensitive rats but not in the resistant strain. Norepinephrine stores were not affected by high salt intake, but tyrosine hydroxylase, and dopamine beta-hydroxylase were elevated in the salt-induced hypertrophied left ventricle in conjunction with increased levels of nerve growth factor and p75 neurotrophin receptor. In contrast, high salt intake reduced ventricular neuropeptide Y in both Dahl salt-resistant and -sensitive rats.

Regulation of sympathetic presynaptic components in rat left ventricle during ligation of abdominal aorta.

To assess the effects of long-term pressure overload on sympathetic presynaptic components in the left ventricle, young adult male rats were subjected to surgical constriction of the suprarenal abdominal aorta. At 4 and 8 wk postsurgery, but not at 1 wk, left ventricular sympathetic activity, measured by the net fractional norepinephrine (NE) decrease after alpha-methyl-p-tyrosine methyl ester administration, was elevated in the aortic-banded rats. However, left ventricular NE was reduced only at 8 wk. Scatchard analysis of saturation binding of [3H]nisoxetine, a specific ligand for NE uptake sites, determined that left ventricular NE transporter sites were also reduced at 8 wk, suggesting a relationship between a reduced number of uptake sites and loss of NE stores. In contrast, aortic constriction did not reduce neuropeptide Y (NPY), tyrosine hydroxylase, dopamine beta-hydroxylase, nervegrowth factor, and low-affinity nerve growth factor receptors at any time point. Thus long-term pressure overload can cause a selective reduction in ventricular NE stores without a reduction in NPY, a colocalized sympathetic neurotransmitter.

Reduced levels of globular and asymmetric forms of acetylcholinesterase in rat left ventricle with pressure overload hypertrophy.

The aim of the present work was to determine the effect of abdominal aortic stenosis on molecular forms of acetylcholinesterase (AChE) in rat heart. Pressure-overload, left ventricular hypertrophy was produced in male Sprague-Dawley rats by suprarenal abdominal aortic constriction. After two weeks the relative heart weight was increased over 20% compared to sham-surgical controls, mostly due to left ventricular enlargement. Aortic constriction reduced AChE activity per wet weight and per unit protein by 25-30% in the left ventricle and interventricular septum, but not in the other chambers. However, total AChE activity per chamber was normal in the left ventricle and interventricular septum, but was elevated in the atria. The molecular forms of AChE were separated in linear sucrose gradients and their specific activities were calculated from the resulting percent activities and total AChE activities. This data showed that although aortic constriction had no effect on ratios of the various forms, it did reduce the specific activities of globular and asymmetric forms in the left ventricle and interventricular septum. The reduced AChE activity suggests that slower rates of ACh hydrolysis occur in the left ventricle in pressure-overload hypertrophy.

Development of neuropeptide Y and tyrosine hydroxylase immunoreactive innervation in postnatal rat heart.

Neuropeptide Y (NPY), immunoreactive (IR), and tyrosine hydroxylase (TH)-IR nerve fibers were scarce at birth in rat heart, but increased rapidly during the first 2 postnatal weeks, reaching approximately adult levels by the third week. The sequence of development was: interatrial septum and atrial wall, free ventricular wall starting from the epicardium, and finally the atrial appendages and interventricular septum. In ventricles and atrial appendages both fiber types developed similarly. In interatrial septum and atrial walls more NPY-IR than TH-IR fibers were evident, and NPY-IR, but not TH-IR, neurons were detected in intrinsic ganglia. Double-label immunohistochemistry provided further evidence that NPY is located in ventricular and atrial noradrenergic nerves, but is also located in nonnoradrenergic nerves in atria.

Glycyl-L-glutamine regulates the expression of asymmetric acetylcholinesterase molecular forms in cultured cardiac post-natal myocytes.

Asymmetric acetylcholinesterase (AChE) forms were associated with pre-natal but not post-natal ventricular myocytes, when myocytes were cultured in a defined medium on laminin-coated plates for 72 h. In contrast, globular AChE molecular forms were associated with both pre-natal and post-natal myocytes. Glycyl-L-glutamine (10(-4) or 109-6) M), but not glycyl-D-glutamine or glycyl-L-glutamate, induced the expression of asymmetric AChE molecular forms by the cultured post-natal myocytes. Neither of the three dipeptides altered the specific activity of cell-associated AChE in the cultured post-natal ventricular myocytes. Tetrameric globular (G4) AChE was the main AChE form secreted by cultured pre-natal and post-natal cardiac myocytes. The secretion rate of AChE from post-natal myocytes was not affected by the addition of glycyl-L-glutamine. These results suggest that glycyl-L-glutamine has a trophic effect on at least one of the components of the post-synaptic cholinergic system in developing rat heart.

Diminished neuropeptide Y and dopamine beta-hydroxylase immunoreactivity in a guinea pig model of left ventricular hypertrophy.

OBJECTIVE: The aims of the study were to determine the effect of chronic pressure overload of the left ventricle on the density and distribution of neuropeptide-Y-like immunoreactive (NPY-LI) nerve fibres in heart and to compare any changes to those observed in adrenergic nerve fibres, identified by dopamine beta-hydroxylase immunoreactivity. METHODS: Pressure overload was produced in female adult guinea pigs by constriction of the abdominal aorta, using a modified Weck haemoclip. The same operation was performed on a separate group of animals except that no clip was placed around the aorta. Five weeks after surgery, animals were anaesthetised, and the hearts were fixed by perfusion for immunohistochemistry. Cryostat sections were stained, using an indirect peroxidase/antiperoxidase method, for NPY or dopamine beta-hydroxylase. RESULTS: Aortic stenosis caused a 45% increase in left ventricular weight and a 58% increase in left atrial weight at 5 weeks postsurgery. Pulmonary oedema, a sign of cardiac failure, was evident in most of the animals with aortic stenosis. Immunohistochemical studies showed that in atria and right ventricles from animals with abdominal aortic stenosis the distribution and density of NPY-LI nerve fibres were similar to those in the sham operated guinea pigs. However, the left ventricles obtained from the animals with aortic stenosis were nearly devoid of NPY-LI nerve fibres. The density of dopamine beta-hydroxylase-LI nerve fibres was also substantially reduced in the hypertrophied left ventricles. CONCLUSIONS: Aortic stenosis resulting in left ventricular hypertrophy caused a nearly complete loss of NPY-LI and dopamine beta-hydroxylase-LI nerve fibres from the left ventricle. The parallel reduction in both neuropeptide Y and dopamine beta-hydroxylase is in accordance with the association of neuropeptide Y with sympathetic (adrenergic) nerve fibres in the left ventricle and suggests that chronic left ventricular hypertrophy causes a severe degeneration of sympathetic axons supplying this chamber and/or reduces the ability of these sympathetic neurones to maintain normal levels of neurotransmitter related enzymes and neuropeptides.

Acetylcholine levels, choline acetyltransferase and acetylcholinesterase molecular forms during thyroxine-induced cardiac hypertrophy.

The effects of left ventricular hypertrophy induced by hyperthyroidism on three biochemical markers of parasympathetic innervation were investigated. In response to subcutaneous injections of thyroxine (400 micrograms/kg; T4) for 6 days, the left ventricle, but not the right, developed significant hypertrophy (20%). In the enlarged left ventricle, acetylcholine (ACh) content and choline acetyltransferase (ChAT) activity per chamber were elevated approx. 25-30%, although no change in these two markers was evident when the data were expressed per unit wet weight. Immunoblot analysis showed that the relative abundance of ChAT protein increased in the hypertrophied left ventricle in correlation with the increased ChAT activity. No changes in ACh content, ChAT activity and ChAT relative abundance were evident in the right ventricle of T4-treated animals. Although hyperthyroidism did not alter AChE specific activity (per unit wet weight) in the left ventricle, the percent activities of the individual AChE globular forms were affected in this chamber. Specifically, T4-treatment reduced the percent activity of globular (G)4 AChE by 20% and increased that of the combined G1 and G2 AChE pool by 15%. Interestingly, in the hypertrophied left ventricle total AChE activity in its extracellular or functionally-relevant pool was reduced due to a loss of G4 AChE activity. These results show that a compensatory increase in parasympathetic innervation can occur during hyperthyroid-induced left ventricular hypertrophy. However, the reduced activity of the functionally-relevant AChE pool suggests that the clearance of ACh after release may be slowed in the hypertrophied left ventricle.

Sympathectomy alters acetylcholinesterase expression in adult rat heart.

STUDY OBJECTIVE: The aim of the study was to determine the significance of adrenergic nerve associated acetylcholinesterase for the pool of total acetylcholinesterase molecules. DESIGN: Acetylcholinesterase was analysed after destruction of adrenergic nerves by 6-hydroxydopamine or bilateral stellate sympathectomy. Effectiveness of treatment was verified by determining noradrenaline concentrations in right ventricle. Acetylcholinesterase activity was assayed in homogenates of atria and portions of left ventricular free wall. SUBJECTS: Adult male Sprague-Dawley rats were used, weight 225-260 g, n = 5 per experimental group. MAIN RESULTS: Sympathectomy caused a small decrease in acetylcholinesterase activity, due to a decrease in the activity of the tetrameric globular form of the enzyme. Choline acetylcholinesterase activity was not altered by sympathectomy, which is an indication that cholinergic nerves were not affected. CONCLUSIONS: The contribution of adrenergic neurones to the cardiac pool of acetylcholinesterase is measurable and consists primarily of the tetrameric globular form of the enzyme.

Changes in the expression of acetylcholinesterase molecular forms during rat heart development.

The development of the molecular forms of acetylcholinesterase was studied in rat heart during the perinatal period. In this study, the activity of acetylcholinesterase increased both per unit wet weight and per unit protein from post-conception day 14 to day 42. Additionally, the activity of atrial acetylcholinesterase per unit wet weight increased more rapidly after birth than that of ventricular acetylcholinesterase. The percent contribution of the various molecular forms to the total acetylcholinesterase pool in heart changed dramatically from the fetal to the neonatal period. This switch primarily consisted of a decrease in the ratio of the asymmetric to globular forms. Thus, the specific activity of globular forms increased while that of the asymmetric forms remained relatively stable. When the atria and ventricles were examined separately at 19 days post-conception, the percent contribution of the individual molecular forms in the two cardiac areas was different. The atria contained a pool of acetylcholinesterase forms similar to postnatal heart while the ventricles contained a pool of acetylcholinesterase forms with a lower globular to asymmetric ratio. Finally, this study showed that greater than half of the acetylcholinesterase pool was inhibited by incubating hearts from fetal rats with echothiopate iodide, suggesting that a large portion of acetylcholinesterase catalytic sites are externalized in fetal heart.

Cardiac acetylcholinesterase molecular forms and choline acetyltransferase in the Dahl (salt-sensitive) hypertensive strain of rats: a regional study.

A regional study of acetylcholinesterase (AChE) molecular forms and choline acetyltransferase (ChAT) in the hearts of Dahl-salt sensitive (DS) and salt resistant (DR) rats was performed in animals administered either 8% or 0.35% dietary NaCl. Atria isolated from DS rats, regardless of dietary NaCl intake, had lower activities of all of the AChE molecular forms and ChAT when compared to their dietary-matched DR controls. In the ventricles, the activities of AChE molecular forms and ChAT were lower in DS rats compared to dietary-matched DR rats only when 8% NaCl diets were administered. The percent contribution of each of the molecular forms to the total AChE pool was not affected by animal strain or diet.

Acetylcholinesterase molecular forms in muscle and non-muscle cells of rat heart.

Experiments were performed to determine the cellular associations of the molecular forms of acetylcholinesterase (AChE) in adult rat heart. For this purpose, a cardiac muscle and a non-muscle fraction were isolated from rat heart ventricles after perfusion with collagenase and hyaluronidase, extracts of these fractions were subjected to ultracentrifugation on linear density gradients of sucrose (5-20%), and fractions of these gradients were analyzed for AChE activity. The results show that only globular AChE molecular forms were present in isolated cardiac muscle cells. Globular AChE forms were also present in the non-muscle cells fraction but in different proportions. The proportions of globular AChE forms plus the high specific activity of choline acetyltransferase in the non-muscle cell fraction suggest that this fraction contains cholinergic nerve fragments. The results of this study also show that asymmetric AChE is released during the perfusion of heart with the digestive enzymes, which suggests that asymmetric AChE is bound to the extracellular matrix of heart.

Regional distribution of the molecular forms of acetylcholinesterase in adult rat heart.

Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, exists as a multiple molecular forms that differ in their quaternary structure and mode of attachment to the cell surface. The distribution of the individual molecular forms of AChE in various cardiac regions with distinct anatomical characteristics was investigated. The results confirmed those of others by showing that the total pool of cardiac AChE had a nonuniform distribution in heart that paralleled the distribution of choline acetyltransferase. The rank order of this distribution was right atrial appendage greater than interatrial septum greater than left atrial appendage = right ventricle = interventricular septum greater than left ventricle. Velocity sedimentation in sucrose gradients of extracts from selected cardiac areas showed that four molecular forms were present in all areas but that the proportions of these forms differed as a function of area. The right and left ventricular walls, the apical portion of the interventricular septum, and the left atrial appendage contained G1 and G4 (globular) AChE in near-equal proportions, but in the basal portion of interventricular septum, the contribution of G4 AChE was greater than that of G1 AChE. The right atrial appendage and the interatrial septum had the largest amount of activity attributable to G4 AChE and the lowest amount attributable to G1 AChE. In all cardiac regions, A12 (asymmetric) AChE comprised 8-10% of the total AChE pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac mitochondrial abnormalities in a mouse model of the fetal alcohol syndrome.

Mitochondrial enzymes and respiration were studied in the hearts of mice exposed to ethanol in utero from gestational Day 8 to parturition. This treatment had previously been shown by electron microscopy to result in myofibril loss and mitochondrial abnormalities. Ethanol was administered to pregnant mice by a liquid diet paradigm and pair-fed dams were used as controls. Ethanol exposure in utero reduced the activities of two mitochondrial inner membrane enzymes, cytochrome c oxidase and succinate dehydrogenase, in the hearts of perinatal mice. Secondly, mitochondrial respiration under both State 3 and 4 conditions with a NAD-linked substrate was depressed in the hearts obtained from the ethanol-exposed fetal mice. However, when a flavin-linked substrate was used, State 3 (ADP-stimulated) but not State 4 respiration was depressed. This study illustrates that in utero exposure to ethanol is deleterious to the functioning of cardiac mitochondria in newborn mice, which in turn could contribute to the development of the heart pathologies present in the Fetal Alcohol Syndrome.

Acetylcholinesterase molecular forms in rat heart.

A comparison of the molecular forms of acetylcholinesterase (AChE) in adult rat atria and ventricles was undertaken. The major forms present in both the atria and ventricles were globular 4S (G1) and 10S (G4) and asymmetric 16S (A12) with minor contributions from 6S (G2) and 12S (A8). Although the total specific AChE activity was higher in atrial samples, no differences in the proportions of the major AChE forms between the atrial and ventricular samples were seen. For example, 16S AChE accounted for 8% to 10% of the total AChE activity in all examined regions of the heart. Cardiac 16S AChE was shown to be soluble in high ionic strength buffer. The addition of EDTA to the extraction buffer resulted in no further solubilization of 16S AChE, indicating that only Type I asymmetric AChE is present in the heart. Additionally, 16S AChE did not require Triton X-100 for extraction. In contrast, 35% of the globular AChE required non-ionic detergent for extraction, which indicates that a percentage of globular AChE in rat heart is membrane-associated.

Maternal ethanol consumption: effect on skeletal muscle development in guinea pig offspring.

The effect of ethanol on developing skeletal muscle was analyzed by examining the gastrocnemius muscle from newborn guinea pigs, exposed to ethanol during the second half of gestation. Electron microscopy revealed vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen and mitochondrial abnormalities in the skeletal muscle samples from the ethanol-exposed newborn guinea pigs. None of these abnormalities were seen in the newborn controls who were born to dams which consumed the same amount of calories as the ethanol-treated dams. The ethanol-associated abnormalities, seen in this study, are similar to those seen in ultrastructural examination of skeletal muscle from chronic alcoholics.

Maternal ethanol consumption: lack of effect on synaptogenesis in layer I of the motor cortex in 19-day-old rat offspring.

Previous studies showed that in utero exposure to ethanol has an adverse effect on development of the central nervous system. Included among those abnormalities is the evidence that in utero exposure to ethanol alters the development of the cerebral cortex in terms of the arborization of pyramidal dendrites and the concentration of neurotransmitters and neurotransmitter receptors. However, limited information is available about the influence of in utero exposure to ethanol on synaptogenesis in the cortex. We examined the influence of chronic maternal ethanol consumption, prior to parturition, on synaptogenesis in layer I of the motor cortex of 19-day-old-rat offspring. Layer I (the molecular layer) was chosen because of the early timing of its development. The motor cortex was chosen because of the evidence of motor problems in children afflicted with the fetal alcohol syndrome. The results of these studies demonstrated that the 19-day-old offspring of rats that were pair-fed, using control or 6.6% (v/v) ethanol-containing liquid diets on a chronic basis prior to parturition, had a comparable density of synapses and distribution of paramembranous density lengths in layer I of the motor cortex as well as a comparable thickness of cortical layer I. Thus, evidence by this and other laboratories, of cortical neurotransmitter and dendritic abnormalities are not reflected in synaptic changes in layer I of the motor cortex at 19 days. It is possible that synaptogenesis is affected at another age or in other regions of the cerebral cortex.

Normal development of cardiac beta adrenoceptors in mice exposed to ethanol in utero.

Abnormalities of cardiac physiology and anatomy may occur in children with the fetal alcohol syndrome. To understand the basis of these abnormalities, research has been performed which shows that in mice exposed to ethanol from gestation day 8 to birth via a liquid diet regime with pairfed controls there are ultrastructural changes in the cardiac myocytes. To determine if the exposure to ethanol also affects the development of the cardiac noradrenergic system, which in turn could cause developmental abnormalities, beta adrenoreceptor binding was characterized by Scatchard analyses of concentration dependent binding curves in newborn mice exposed to ethanol in utero. Although body and heart weight were lower in newborns from both the pairfed and ethanol groups compared to a normally fed group, no differences in the densities of the beta adrenoceptors or in the Kd values for binding were seen due to the ethanol or the liquid diet regime. Therefore, at least one component of the development of the cardiac sympathetic system was not altered by ethanol exposure or a reduced caloric intake.

A possible role for beta-adrenergic receptors in the expression of audiogenic seizures.

DBA/2 mice are genetically prone to audiogenic seizures and, when compared with seizure resistant C57BL/6 mice, were found to have an increased density of beta-adrenergic receptors in their midbrain at the age of peak seizure susceptibility. Propranolol, a beta-receptor blocking agent, attenuated all stages of the seizure syndrome. However, a comparison of the effects of its d- and l-isomers suggested that propranolol's anticonvulsant activity was due to its local anesthetic-like action. Pindolol, a more potent beta blocker that is at least 100 times less potent than propranolol with respect to local anesthetic-like activity, produced anticonvulsant effects in approximately the same systemic dose range as propranolol. This indicates that pindolol's anticonvulsant activity could be due to beta blockade and, taken together, these data suggest that beta-adrenergic receptors may play a role in the expression of audiogenic seizures in these animals.

Inhibition of synaptosomal enzymes by local anesthetics.

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na+ + K+)-ATPase (ouabain-sensitive), Mg2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPases, for example 7.9 and 10 mM tetracaine for Mg2+-ATPase and (Na+ + K+)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.