THE DISTRIBUTION AND METABOLISM OF PUTRESCINE, SPERMIDINE AND SPERMINE INJECTED INTO THE CEREBRAL VENTRICLES OF RABBITS

Following the intracerebroventricular injection into rabbits of spermidine or spermine the highest concentrations were initially found in the caudate nucleus, hypothalamus and medulla. Subsequently there was a rapid decline in the amounts present in the caudate nucleus and hypothalamus and, particularly in the case of spermidine, an increase in the conccntration in the lower brain stem and cervical cord. This pattern of changes is consistent with the amines being redistributed by passage in CSF. Intraventricularly injected putrescine followed the same initial distribution pattern but within 2 days it had been largely converted to spermidine and spermine. Synthesized polyamines accumulated in all the regions examined. The time course of synthesis indicated that spermidine was the precursor of spermine. Spermine was also formed from injected spermidine and vice-versa. These findings concur with the pharmacological and neurotoxic actions of putrescine, spermidine and spermine.     

CLEARANCE OF THE POLYAMINES FROM THE PERFUSED CEREBROVENTRICULAR SYSTEM OF THE RABBIT

Abstract— The clearance of the polyamines spermidine and spermine from cerebrospinal fluid was investigated in the rabbit by ventriculocisternal perfusion. Clearance involved both saturable and nonsaturable uptake processes. The saturable component was a high affinity system with an affinity constant of 21 μ m for spermidine and 24 μ m for spermine. The clearance of spermidine was reduced by the presence of spermine and vice-versa. Other polyamine congeners also reduced spermidine and spermine clearance and it is suggested that the two polyamines share the same carrier. Evidence for concentrative uptake of polyamines into choroid plexus is presented and it is suggested that an active system may also transport polyamines into brain tissue. At high perfusion concentrations simple diffusion may also take place.     

THE BIOCHEMICAL ROLE OF NATURALLY OCCURRING POLYAMINES IN NUCLEIC ACID SYNTHESIS

1. The polyamines, putrescine, spermidine and spermine occur in free or acetylated form in a wide variety of living organisms. Putrescine is biosynthesized from ornithine or arginine; spermidine and spermine from methionine and either ornithine or arginine. 2. It is difficult to determine the intracellular distribution of polyamines since they are all very soluble in water and they are readily redistributed when cells are disrupted. Evidence suggests that a substantial proportion of the intracellular polyamines is attached to the ribosomes and that spermidine is not concentrated in the nucleus. 3. Polyamines bind strongly to both DNA and RNA. The strength of binding is:spermine > spermidine > putrescine. Polyamines stabilize the double helix of DNA, probably by forming a bridge across the narrow groove, by involving electrostatic bonding with the phosphate group. However, they do not appear to alter the overall conformation of DNA. Spermine enables single-stranded RNA to fold into a more compact configuration which is less susceptible to attack by ribonuclease. 4. Spermine and spermidine are able to stimulate the DNA primed RNA polymerase. They facilitate the removal of RNA from the DNA-RNA-enzyme complex. 5. Polyamines promote the association of ribosomal subunits and also the binding of amino acyl transfer RNA to ribosomes. They cause increased coding ambiguities in the process of translation in certain bacterial systems. 6. There is a close correlation between the intracellular concentration of spermidine and the rate of RNA synthesis both in rat liver and in Escherichia coli. Conditions which affect the rate of RNA synthesis also affect the concentration of free intracellular spermidine. 7. Bacteria usually contain putrescine and spermidine, whereas animal tissues contain spermine and spermidine. Spermidine probably fulfils the same role in both bacteria and animal tissues, but the presence of spermine, which is common to eucaryotes, is possibly associated with their more complex mechanisms for regulating RNA and protein synthesis.     

POLYAMINE METABOLISM IN THE BRAIN AND LIVER OF THE DEVELOPING MONKEY

Abstract— The concentrations of putrescine and the polyamines, spermidine and spermine, along with the specific activities of the enzymes involved in their biosynthesis, ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase have been measured in brain and liver of the developing Rhesus monkey from mid-gestation, through birth and neonatal life to maturity. The results suggest that it is an increased concentration of putrescine and an increased specific activity of ornithine decarboxylase which are associated with the rapid growth of fetal brain during the middle of gestation. By the end of two-thirds of gestation, both of these parameters have attained values similar to those found in mature brain. The concentration of spermidine in brain and the specific activities of S -adenosylmethionine decarboxylase and spermidine synthase are lower in fetal brain than adult brain and increase slowly after birth to reach values similar to those of the adult only after several months. These results provide additional evidence that in the mature brain spermidine serves some function other than one associated with rapid growth.
Fetal liver at mid-gestation was characterized by increased concentrations of both putrescine and spermidine and increased specific activities of the enzymes which synthesize them. By two-thirds of gestation, values similar to those found in adult liver had been attained. Liver has thus reached maturity with regard to polyamine metabolism by this time.     

Acetylation of polyamines in chicken brain and retina

Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration.     

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

REGIONAL DISTRIBUTION OF PUTRESCINE, SPERMIDINE AND SPERMINE IN RELATION TO THE DISTRIBUTION OF RNA AND DNA IN THE RAT NERVOUS SYSTEM

—Putrescine, spermidine, spermine, RNA, DNA and protein concentrations were determined in 14 parts of the rat nervous system. If the concentrations are expressed in DNA units, putrescine and spermidine concentrations change concomitantly in the different brain parts, with the exception of hypothalamus, where relatively higher putrescine than spermidine concentrations are observed. The constancy of putrescine/spermidine ratios indicates the value of putrescine concentration as an index of spermidine biosynthesis. Spermidine correlates with RNA, except in medulla, spinal cord and peripheral nerves. It is assumed that the relative excess of spermidine in these structures indicates an additional functional role. Spermine/DNA ratios are remarkably constant in the diencephalic and telencephalic regions; they are also nearly constant, but significantly lower in midbrain, medulla, spinal cord and cerebellum. This observation gives additional support for the preferential interrelation of spermidine with RNA and spermine with DNA; i.e. for different functional roles of these two narrowly related polycations.     

Alterations in the accumulation patterns of polyamines in brains of myelin-deficient mice.

Quaking mutants and jimpy mutants of mice have known deficiencies of myelination of the central nervous system, as well as lesser involvement of the peripheral nervous system. Both mutants also have altered polyamine synthesis and accumulation, particularly in the hindbrain and spinal column. The ratio of spermidine/spermine, which generally is higher in tissues with high rates of biosynthetic activity, was significantly lower in the mutants as compared to their normal siblings. In quaking mutants, 5 months of age, the spermidine concentration of hindbrain and spinal column was 60% that of controls. In contrast, the decreased spermidine/spermine ratio in jimpy mutants resulted from a marked increase in the spermine concentration in both forebrain and hindbrain. Alterations in the spermidine/spermine ratio could lead to reductions in the biosynthetic potential of the brain during development.     

Alterations in the accumulation patterns of polyamines in brains of myelin-deficient mice

Quaking mutants and jimpy mutants of mice have known deficiencies of myelination of the central nervous system, as well as lesser involvement of the peripheral nervous system. Both mutants also have altered polyamine synthesis and accumulation, particularly in the hindbrain and spinal column. The ratio of spermidine/spermine, which generally is higher in tissues with high rates of biosynthetic activity, was significantly lower in the mutants as compared to their normal siblings. In quaking mutants, 5 months of age, the spermidine concentration of hindbrain and spinal column was 60% that of controls. In contrast, the decreased spermidine/spermine ratio in jimpy mutants resulted from a marked increase in the spermine concentration in both forebrain and hindbrain. Alterations in the spermidine/spermine ratio could lead to reductions in the biosynthetic potential of the brain during development.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

INTERRELATIONS BETWEEN POLYAMINES AND NUCLEIC ACIDS: CHANGES OF POLYAMINE AND NUCLEIC ACID CONCENTRATIONS IN THE DEVELOPING RAT BRAIN

The rat brain concentrations of protein, RNA, DNA, putrescine, and of the polyamines spermidine and spermine, were studied during development. Putrescine formation is apparently controlled by ornithine decarboxylase. Spermidine and spermine concentrations change in inverse directions to their anabolic enzymes. It has been presumed, therefore, that the low concentrations of the polyamine-synthesizing enzymes in immature brain are compensated for, by high putrescine and S-adenosylmethionine concentrations. In agreement with previous findings for fish brain, the changes in RNA and spermidine concentrations were most closely correlated. The functions of DNA: spermine are directly correlated only during the periods of brain maturation, after cell proliferation has nearly ceased.     

INTERRELATIONSHIPS BETWEEN POLYAMINES AND NUCLEIC ACIDS. CHANGES OF POLYAMINE AND NUCLEIC ACID CONCENTRATIONS IN THE GROWING FISH BRAIN (SALMO IRIDEUS GIBB.)

Abstract— In contrast to mouse brain, the content of putrescine in fish brain considerably exceeds that of spermine and spermidine. While we observed constant protein, RNA and spermidine concentrations in fish brains of weights between 60 and 800 mg, DNA and spermine concentrations diminished with increasing brain weight, the content of spermine per cell being constant throughout life. It can be concluded from our results that growth of fish brain results both from cell enlargement and cell proliferation. The concomitant changes of spermine and DNA concentrations in the growing fish brain are the first example of a direct quantitative relationship between these cell constituents and provides evidence on their possible functional relationship in the cell nucleus.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

Circadian variation in cerebral spermine and spermidine concentrations in entrained and free-running mice

In the present study, although some evidence in favour of a circadian variation in brain spermidine concentration was obtained in Swiss mice, peak concentrations occurred during darkness and were therefore out of phase with those reported in a previous study in the same mouse strain (Rodichok and Friedman, 1978). In addition the largest magnitudes of oscillation (34.8 and 22.7% for spermidine and spermine, respectively) were only about one quarter as large as those previously reported. The changes in polyamines did not correlate with change in locomotor activity. In C.D. mice a more complex pattern of change in spermidine was observed with peaks occurring in both the light and the dark phases of the cycle. Exposure of these animals to continuous light or continuous darkness modified the pattern of these fluctuations but did not abolish them.

It is concluded that, although daily fluctuations in spermidine and spermine concentration do occur in both entrained and free running mice such changes do not correlate with the phase of the light-dark cycle or with locomotor activity.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Polyamines Stimulate Mitochondrial Calcium Transport in Rat Brain

The effects of the polyamines spermine and spermidine on rat brain mitochondrial calcium transport were examined using a variety of techniques for measuring the kinetics of calcium uptake and the buffering capabilities of isolated mitochondria. Spermine both increased the rate of calcium accumulation and decreased the set-point to which isolated mitochondria buffer free calcium concentration. In the presence of physiological concentrations of sodium and magnesium, spermine lowered the extramitochondrial calcium level to approximately 0.3 microM, a value close to the resting intracellular calcium concentration. The effect of polyamines was concentration dependent, with a half-maximal effect of spermine observed at approximately 0.1-0.4 mM (respiratory substrate dependent), whereas spermidine was approximately 10 times less potent. Calcium transport by hippocampal mitochondria was stimulated markedly more by spermine than was calcium transport by mitochondria isolated from brainstem. The stimulatory effect of spermine was not due to an increase in the transport of respiratory substrates inside the mitochondria nor to an effect on the enzymes using these respiratory substrates. An examination of the effect of spermine on the kinetics of calcium uptake indicated that spermine increased calcium uptake maximally at low calcium concentrations. Beyond that level, the stimulatory effect of spermine decreases, and spermine can even inhibit calcium uptake. These results are in good agreement with previous reports on the effects of polyamines on calcium transport in mitochondria from peripheral tissue. They support the hypothesis that spermine increases the rate of calcium uptake by mitochondria by increasing the affinity of the uniporter for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamines and amino acid incorporation in vitro into microsomes of rat cerebral cortex

1. Polyamines were found to be associated with microsomes of rat cerebral cortex, the amount of spermine being about four times that of spermidine. Cell sap contained more spermidine than spermine. 2. Both polyamines were able to stimulate the incorporation of [(14)C]valine into microsomes in vitro with a maximum rate equal to 250% of the control. Polyamines stimulated at concentrations close to the amount of spermine and spermidine naturally present in the system. 3. Spermine (0.05mm) was used to study the mechanism of action of polyamines. The increasing of microsome and cell-sap concentration facilitated the action of spermine, but the same process was inhibited by increasing pH5-enzyme concentration. 4. Spermine did not affect the association of [(14)C]valine with tRNA in cell sap, but increased the rate of aminoacyl-tRNA formation in pH5 enzyme preparations. However, this process was not affected in any case when incorporating microsomes were present. 5. It is suggested that microsomes are the main site of action of polyamines.     

Changes in Polyamine Concentrations in Amygdaloid-Kindled Rats

Concentrations of the polyamines putrescine, spermidine, and spermine were investigated in the left and right amygdala and in the remaining cerebrum, in which kindling was induced by repeated application of electrical stimulation of the left amygdala of rats. In kindled rats, the concentrations of spermidine and spermine increased slightly, but elevations did not reach significant levels in any brain regions. The most profound increase was detected in the putrescine concentration in all parts of the cerebrum 1-8 h after the final stimulation. These results suggest that the increases in concentrations of polyamines, particularly of putrescine, are involved in the pathogenesis of amygdaloid kindling.     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.