Hypertension in normotensive and hypertensive rats by spermine ingestion.

Polyamines (putrescine, spermidine and spermine) play an important role in the development of hypertension and in the expression of atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of blood pressure. Wistar Kyoto normotensive (WKY) and spontaneously hypertensive rats (SHR) were given spermine in drinking water (0.5%) for 15 days. The spermine intake elevated the blood pressures of both SHR and WKY rats and reduced the expression of ANP (Northern blotting) in the ventricles. ANP levels in the plasma determined by enzyme immunoassay (EIA) showed no changes in the levels of plasma ANP after spermine intake. An analysis of polyamines by high-pressure liquid chromatography showed that the levels of spermine and spermidine were elevated in SHR hearts. It was in SHR hearts alone that spermine intake was associated with increases in the levels of putrescine. The results suggest that spermine-induced increases in blood pressure may involve mechanisms other than ANP.

Attenuation of isoproterenol-mediated myocardial injury in rat by an inhibitor of polyamine synthesis.

OBJECTIVE: Ornithine decarboxylase (ODC) is an initial rate-limiting enzyme in the synthesis of polyamines (putrescine, spermidine, and spermine) that play a role in cell growth and differentiation. Recent studies have shown that spermidine and spermine cause injury to a variety of cells including myocytes in vitro. In this investigation, we used alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC activity and polyamine synthesis to test the hypothesis that polyamines contribute to myocardial injury in rat. METHODS: Male Sprague Dawley rats were treated with (i) saline (0.2 ml/day, s.c.), (ii) isoproterenol (ISO) (5 mg/kg/day for 8 days, s.c.) to produce necrotizing myocardial injury, or with (iii) DFMO + ISO. DFMO was started 2 days before the initiation of ISO and both ISO and DFMO were continued until the end of the experimental period. Myocardial injury was assessed by determining the increased release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) into the plasma, and by morphometric analysis of the lesion area in heart sections stained with Gomori trichrome. RESULTS: ISO induced the release of CPK and LDH by 6 hr and 24 hr, respectively, and produced subendocardial necrosis, which was both acute and resolving following 8 days of ISO. DFMO treatment inhibited ISO-induced increases in (i) ODC activity and putrescine and spermidine levels in heart, (ii) CPK and LDH activity in plasma, and (iii) the area of subendocardial lesions. CONCLUSIONS: These observations suggest that polyamines are one of the intracellular factors that contribute to ISO-mediated cardiac injury in the rat.

Mechanism of polyamine toxicity in cultured cardiac myocytes.

The goal of this study was to investigate the mechanism of polyamine-mediated injury to the cardiac myocytes isolated from neonatal rat hearts. The myocytes, cultured in Dulbecco's minimal essential medium-1% foetal calf serum (FBS), were exposed to spermidine or spermine. The toxicity to myocytes was determined by (a) increased release of creatine kinase (CPK) into the media and (b) decline in cell viability or functional activity. Spermidine, above 10 mum, increased the release of CPK into media, decreased cell viability and decreased the functional activity of the myocytes. The FBS exhibited polyamine oxidase activity and semicarbazide-sensitive amine oxidase activity. Aminoguanidine, MDL72,527 or semicarbazide, are the inhibitors of amine oxidases, polyamine oxidase (PAO) and semicarbazide-sensitive amine oxidase (SSAO), respectively. The addition of these inhibitors to the medium protected the myocytes from spermidine toxicity. To determine whether myocyte PAO is involved in polyamine toxicity, we used horse serum that contained high SSAO activity and negligible PAO activity. The myocyte extracts had negligible SSAO activity but high PAO activity. When myocytes were cultured in horse serum in lieu of FBS, spermine caused toxicity at above 100 mum. In horse serum, MDL72,527 and semicarbazide protected the myocytes from spermine toxicity. These observations show that extracellular amine oxidases and myocyte PAO are significant in mediation of polyamine toxicity.

Differential induction of polyamine oxidase activity in liver and heart of iron-overloaded rats.

The present study was undertaken to investigate the effect of iron dextran treatment on polyamine oxidase (PAO) activity, iron accumulation, and lipid peroxidation in livers and hearts of rats. PAO catalyzes oxidative deamination of polyamines, the cellular aliphatic cations. This reaction produces highly toxic hydrogen peroxide, 3-acetamidopropanal, and precursors of higher polyamines. The rats were given iron dextran daily for 7 d. In iron-dextran-treated rats, a marked increase in the hepatic level of iron was associated with enhanced lipid peroxidation and increased PAO activity. Though iron accumulation and lipid peroxidation in the iron-treated rats increased significantly in the heart, PAO activity remained unchanged. The paraffin sections of livers stained with Perls iron stain showed the presence of iron in macrophages and hepatocytes. The sections of hearts showed iron deposits only in macrophages, while myocytes showed no iron staining. These results show that although iron dextran treatment results in accumulation of iron in both liver and heart, it induces PAO activity only in liver. The significance of increased PAO activity in lipid peroxidation and fibrosis in iron-mediated injury is discussed.

Polyamine regulation of atrial natriuretic peptide in cultured cardiocytes.

In the following investigation, we have studied the role of polyamines in the regulation of atrial natriuretic peptide (ANP) using ventricular cardiocytes which in culture synthesize and secrete ANP. Polyamines are cellular cations ubiquitous in eukaryotes, and ANP is a hormone synthesized and secreted by the cardiac atria in adult animal. The cardiocytes were isolated from neonatal rat hearts by enzymatic dissociation using trypsin and collagenase. The functional role of polyamines in regulation of ANP was assessed by exposing the cardiocytes to difluoromethylornithine (DFMO) which is an inhibitor of ornithine decarboxylase, an initial rate-limiting enzyme in the biosynthesis of polyamines. The results showed that DFMO reduced the levels of putrescine (diamine) and spermidine (triamine) in cultured cardiocytes, and it decreased the levels of ANP in media and cellular extracts of cardiocytes as a function of its dose. An addition of putrescine (100 microM) restored within 5-15 min the levels of ANP in media of both control and polyamine-depleted cardiocytes. These results suggest that polyamines are one of the cellular factors involved in regulation of ANP secretion in cultured cardiocytes.

Effect of difluoromethylornithine on atrial natriuretic peptide in rat atria.

Polyamines are aliphatic cations known to have a role in cellular growth and differentiation. In this study, difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, was used to investigate its effect on atrial natriuretic peptide (ANP) in atria of rat. The rats were given DFMO (2%) in drinking water for 10 days and three intraperitoneal administrations (200 mg/kg), 24, 18 and 1 h prior to experiment. Radioimmunoassay of ANP in atrial extracts indicated that DFMO treatment increased ANP contents of atria. ANP from atrial extracts was immunoprecipitated using ANP antibody and the immunoprecipitate was resolved and detected by SDS-PAGE and Western blotting. The predominant ANP peptide in both control and DFMO group migrated at 17 kDa. The synthesis of ANP was studied following the intravenous administration of [35S]methionine. ANP in atrial extracts was immunoprecipitated by ANP antibody. The incorporation of ANP into control and DFMO group peaked at 30 min and returned to a basal level by 60 min. DFMO decreased the incorporation of [35S]methionine into ANP. At 30 min following the administration of [35S]methionine, putrescine restored the synthesis of tricholoroprecipitable atrial proteins, but had no effect on the synthesis of ANP. At 60 min following, the amount of labeled ANP in DFMO + putrescine-treated group was significantly lower than that in DFMO group. These results indicate that polyamines influence both the synthesis and secretion of ANP.

Purification and characterization of semicarbazide-sensitive amine oxidase from porcine aorta.

We purified semicarbazide-sensitive amine oxidase (SSAO) from porcine aorta by sequential DEAE-Sephacel, DEAE-Sephadex and Affi-gel-Con A chromatography. The analysis of this protein under denaturing conditions exhibited two protein bands migrating at 110-107 kDa. Under non-denaturing conditions only a single protein band was observed. By isoelectric focusing pI of SSAO was estimated to be 5.5. The apparent Km and Vmax of porcine SSAO for oxidation of benzylamine were 4.5 microM and 200 nmol/hr/mg protein, respectively. Porcine SSAO was inhibited both by semicarbazide and phenelzine while deprenyl or clorgyline were without any effect on enzyme activity. IC50 for inhibition of semicarbazide and phenelzine was 0.015 microM and 1 nmol, respectively.

Inhibition of polyamine synthesis impairs the secretion of atrial natriuretic peptide.

The aim of the present study was to evaluate in vivo the role of polyamines in the secretion of atrial natriuretic peptide (ANP). alpha-Difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase activity and polyamine synthesis was given in drinking water and through intraperitoneal administration to Sprague-Dawley rats. Carotid artery was cannulated for collection of blood samples and measurement of blood pressure following the administration of arginine-vasopressin (AVP). Analysis of polyamines in cardiac tissue indicated that DFMO treatment decreased contents of putrescine and spermidine in cardiac tissue by 80% and 48%, respectively. Quantitation of ANP in plasma by radioimmunoassay indicated that both basal and stimulated levels of ANP in DFMO-treated animals were 21.5% and 50% of those in control rats. The administration of putrescine restored the levels of basal and AVP-stimulated levels of ANP in plasma which confirmed that DFMO effect on ANP secretion occurred specifically through the polyamine pathway.

Phosphorylation of ornithine decarboxylase by casein kinase II from RAW264 cells.

Casein kinase II and ornithine decarboxylase were purified from a virally-transformed macrophage-like cell line, RAW264. The addition of casein kinase II to a reaction mixture containing [tau-32P]GTP, Mg++, and ornithine decarboxylase led to the phosphorylation of a 55,000 dalton protein band in the purified preparation of ornithine decarboxylase. Stoichiometric estimates indicated that casein kinase II incorporated 0.15 mole of phosphate per mole of ornithine decarboxylase, which was increased to 0.3 mole/per mole in the presence of spermine. The apparent Km and Vmax values for the casein kinase II-mediated phosphorylation of ornithine decarboxylase were 0.36 microM and 62.5 nmol/min./mg kinase. The addition of spermine to the reaction did not alter the Km but increased the Vmax to 100 nmol/min./mg kinase. The phosphorylation of ornithine decarboxylase by casein kinase II affected neither the rate of maximal ornithine decarboxylase activity nor the affinity of the enzyme for ornithine.

Immunolocalization of ornithine decarboxylase in rat heart atria.

Mammalian atrial myocytes secrete a potent natriuretic and diuretic factor, atrial natriuretic peptide (ANP), in response to volume expansion or elevations of right atrial pressure. ANP is synthesized in the atrial atrial myocytes and stored in cytoplasmic secretory granules. Ornithine decarboxylase (ODC) is the initial rate-limiting enzyme in the biosynthesis of polyamines, organic cations which are essential for various aspects of DNA, RNA and protein synthesis, structure, and function. The enzyme has been reported to be localized predominantly in the cytoplasm. A polyclonal antibody elicited against ODC was used to analyse the intracellular distribution of the enzyme protein within atrial myocytes at the ultrastructural level through the use of a post-embedding immunogold technique. In control animals, the density of gold particles associated with the atrial granules was seven to 30-fold higher than that detected in association with other subcellular structures. Administration of Isoproterenol increased atrial of Isoproterenol increased atrial ODC activity 18-fold and the density of the immunolabelling of the atrial myocytes five-fold. Statistically significant increases in the density of labelling after stimulation occurred in association with the atrial granules and the nucleus. After isoproterenol stimulation, 60% of the immunodetectable ODC protein in the atrial myocyte was found in association with the atrial granules. The atrial granules were demonstrated to contain ANP by immunocytochemical analysis of adjacent sections.

Isoprenaline induced changes in ornithine decarboxylase activity and polyamine content in regions of the rat heart.

Isoprenaline stimulated increases in ornithine decarboxylase activity and polyamine content in cardiac tissues are implicated in macromolecular synthesis and cellular growth, but little is known about polyamine metabolism in functionally distinct regions of the heart. We therefore determined regional changes in ornithine decarboxylase activity and polyamine content in the right and left ventricles, septum, and the right and left atria of the rat following the administration of isoprenaline. An increase in ornithine decarboxylase specific activity and tissue polyamine content occurred in all cardiac regions, but the highest ornithine decarboxylase activity was found in the septum. Propranolol inhibited the isoprenaline stimulated ornithine decarboxylase activity in all the regions. Putrescine increased and peaked between 6 and 8 h in right and left ventricles and septum and declined to a control level by 12 h. Following a peak increase at 8 h, spermidine and spermine contents of both ventricles were maintained at peak levels, while those in the septum declined to control values by 12 h. There was no detectable putrescine in the right atria from the control experiments and in either atrium at 2 h following isoprenaline administration. Putrescine content peaked at 6 h in the right atrium and at 8 h in the left atrium and then declined. In both atria there was a peak increase in spermidine and spermine contents between 4 and 8 h. These results show that there is a regional variation in the accumulation of polyamines in the rat heart following isoprenaline administration.

Mucosal ornithine decarboxylase in the small intestine: localization and stimulation.

In most tissues increases in ornithine decarboxylase (ODC) are associated with growth. Refeeding fasted rats. a potent stimulus for mucosal growth, strongly increases ODC in both small and large intestinal mucosa. In the small bowel, almost all of this increase occurs in the mature villus cells rather than the proliferative crypt cells. Nevertheless, inhibition of ODC with difluoromethylornithine blocks the growth response. Using a highly specific, polyclonal antiserum to ODC, we have determined that in the fasted rat ODC is localized almost exclusively to the villus cells. Using antiserum dilution techniques, we have shown that, within 2 h, refeeding increases the amount of immunoreactive ODC in both villus and crypt cells. Furthermore, the trophic hormone gastrin also increases ODC, but only in the crypt cells. Epidermal growth factor increased ODC to a greater extent than gastrin, but stimulation was more general, including both crypt and villus cells. Perfusing an isolated segment of small bowel in situ with glycine for 2 h also increased immunoreactive ODC but only in the villus cells. Thus in the small intestine the effect of refeeding on ODC activity appears to be mediated by different types of stimuli: luminal nutrients increase enzyme levels in the absorbing villus cells, while trophic peptides stimulate ODC synthesis in the proliferative crypt cells.

Regional changes in ornithine decarboxylase activity and polyamine levels during thyroxine-induced cardiac hypertrophy.

Ornithine decarboxylase activities (ODC) and polyamine levels were determined in five cardiac regions of the rat heart, following daily administration of 1 mg/kg of thyroxine, in the right and left atria, the right and left ventricles and the septum. The thyroxine stimulated ODC activity in all five regions of the heart. Enzyme activity in the left atrium and the septum peaked a day earlier than in other regions and the decline of ODC activity was slower. Putrescine in control animals was present in all regions except the right atrium, where its content was below detectable levels. Following the administration of thyroxine, the putrescine content of the left atrium, the right ventricle and the septum declined, while spermidine and spermine levels remained unchanged. In direct contrast to the other regions of the heart, thyroxine stimulated an increase in polyamines, as well as in weight which occurred exclusively in the left ventricle. These findings suggest a causal relationship between increased polyamines and hypertrophy.

Characterization of casein kinase II from a virally transformed macrophage-like cell line, RAW264.

Casein kinase II from a virally-transformed macrophage cell line (RAW264) was purified by a sequential DEAE, Procion Red, phosvitin-Sepharose and heparin-Sepharose chromatography. With [tau-32P]GTP as a phosphate donor and casein as a substrate, the kinase was stimulated by polyamines and inhibited by heparin. The purified kinase had a specific activity of 1137 nmol/min/mg protein and exhibited three major protein bands of 40 K, 35 K, and 25 K. Under non-denaturing conditions in 50 mM Tris-50 mM NaCl the enzyme was eluted as a single peak with molecular weight of 110 K. Incubation of kinase in the presence of [tau-32P]GTP and Mg2+ resulted in phosphorylation of the 25 K protein band of the enzyme. In the presence of [tau-32P]GTP and Mg2+ the kinase was able to phosphorylate 55 K protein band in purified ornithine decarboxylase preparation from RAW264 cells and the rat-type II regulatory subunit of the cyclic AMP-dependent protein kinase.

Gastric mucosal ornithine decarboxylase: localization and stimulation by gastrin.

Gastrin injection and refeeding fasted rats are effective trophic stimuli for the oxyntic gland mucosa of the stomach. Neither stimulus increases detectable ornithine decarboxylase (ODC) activity in the tissue. Difluoromethylornithine (DFMO), a potent inhibitor of ODC, blocks the mucosal growth response, indicating that ODC activity is necessary for growth. Elevated levels of spermidine and spermine are detectable in the mucosa after gastrin administration. Using a highly specific, polyclonal antiserum to ODC, we determined that the enzyme is present in oxyntic gland mucosa confined to a narrow band of cells at the base of the gastric pits and openings of the glands. In antral mucosa, ODC is present throughout the lower 20% of the mucosa, which consists of the necks and pyloric glands. Using antiserum dilution techniques, we show that gastrin administration increases immunoreactive ODC in the oxyntic gland area but not in the antral mucosa, where it has no trophic effect. Elevated cellular content of ODC is apparent within 2 h after injection of gastrin, peaks at 4 h, and declines to basal levels by 12 h. Gastrin-stimulated increase in ODC is confined to the narrow band of cells in which low levels of the enzyme protein were detected in control animals. The decarboxylating activity detectable in oxyntic gland mucosal extracts is not inhibited by administration of DFMO or cycloheximide, each of which inhibits ODC activity in other tissues. Addition of unlabeled lysine to the decarboxylation assay reaction of oxyntic gland mucosa extract inhibits the decarboxylation of radiolabeled ornithine substrate. Thus it is likely that the stomach possesses nonspecific decarboxylase activity, which accounts for most of the measured activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis and transport of gangliosides in peripheral nerve.

Radiolabelled glucosamine was injected into L-7 dorsal root ganglion (DRG) of rabbits. At several different times after injection DRG, lumbosacral trunks (LST) and sciatic nerves (SN) removed and gangliosides extracted. Two and 3 weeks after injection the amounts of radioactivity in the ganglioside fractions of LST SN were significantly higher than at days 1 and 2. The TCA soluble radioactivity decreased dramatically over the same time period. Colchicine prevented the appearance of radiolabelled lipid in LST and SN. From these experiments we conclude that some ganglioside is synthesized in the neuronal cell bodies of DRG and transported in the axons of the sciatic nerve. In another experiment the sciatic nerve was transected and ends separated to prevent regeneration. Ganglioside synthesis and transport were studied in these animals the same way as the previous experiment. There was no difference the amount of radiolabelled ganglioside that was isolated from DRG or LST of transected compared with control nerves. The behavior of several potential acid soluble contaminants was studied in several steps used to isolate gangliosides. Of those studied only CMP-NeuAc could cause significant contamination of the final ganglioside preparation.

A correlation of the alpha-bungarotoxin binding sites (acetylcholine receptors) and intramembranous particles in denervated skeletal muscle of rat.

A quantitative distribution of alpha-bungarotoxin (alpha-BGT) binding sites i.e. acetylcholine receptors (AchR) has been investigated and correlated with the distribution of intramembranous particles (approximately 15 nm in diameter) observed on the freeze-fractured face (P-face) of the non-synaptic sarcolemma of denervated muscle. By both light and electron microscopy autoradiography, randomly distributed clusters of alpha-BGT sites are visualized on the plasma membrane of the denervated muscle. Such distributions of the toxin binding sites correspond with that of the 15 nm particles on the P-face of the denervated muscle. Quantitative studies suggest that upon denervation the toxin binding sites increase approximately 60-fold in the non-synaptic sarcolemma. However, the density of these alpha-BGT sites is 4--5 times more than the density of the 15 nm particles. On the basis that there are two alpha-BGT binding sites per AchR molecule, these results suggest that each 15 nm particle is composed of more than one AchR receptor. In addition to the increase in toxin binding sites on the non-synaptic sarcolemma, a notable increase in the number of silver grains is observed in the peripheral sarcoplasm and is speculated to be part of the intracellular pool of receptors.

alpha-bungarotoxin binding sites (acetylcholine receptors) in denervated mammalian sarcolemma.

The nonsynaptic sarcolemma of denervated skeletal muscle of rat shows an abundance of approximately 15 nm intramembranous particles on the P face. These particles are either singly distributed or are in clusters, and they are essentialy lacking from the comparable freeze-fractures of the innervated sarcolemma. Autoradiographic studies using 125I-alpha-bungarotoxin (BGT) on 1 mu-thick sections, and freeze-etch studies using ferritin-alpha-BGT conjugates on membrane fractions, show that the distribution of the label corresponds to the distribution of the 15-nm particles in the nonsynaptic sarcolemma. On the basis of these results and existing physiologic and biochemical data, it is suggested that the 15-nm intramembranous particles are components of the alpha-BGT binding sites, ie, acetylcholine (Ach) receptors, in the nonsynaptic sarcolemma of denervated muscle and that the two types of distributions represent two spatial manifestations of Ach receptor molecules. The significance of these findings in relation to synapse formation in denervated muscle is discussed.