Regulatory interrelations between GABA and polyamines. I. Brain GABA levels and polyamine metabolism

Elevation of brain GABA levels by GABA-T inhibition is accompanied by a decrease ofS-adenosylmethionine decarboxylase activity. This is followed by an increase of ornithine decarboxylase activity and a severalfold increase of brain putrescine levels. Spermidine and spermine levels are not significantly affected under these conditions. These unexpected findings support a regulatory interaction between GABA and polyamine metabolism.     

Inhibition of polyamine metabolism

Book Review

Inhibition of polyamine metabolismP.P. McCann, A.E. Pegg and A. Sjoerdsma (Eds), San Diego: Academic Press Inc. 1987. xvi + 371 pages. $75. ISBN 0-12-481835-8     

Selective regulation of polyamine metabolism with methylated polyamine analogues

Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N ¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.     

Glutamate and GABA receptors in vertebrate glial cells

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.     

Calcium metabolism in vertebrate photoreceptors

So far all attempts to demonstrate a rapid, light-stimulated release of calcium from disks into the cytosol at a sufficiently high stoichiometry have failed. Either the release stoichiometry was too small or the velocity too slow to account for the amplification in visual transduction. The multitude of failures demonstrate that regulation of intracellular calcium is a very delicate process and the idea of a robust calcium channel in the disk membrane that is opened by rhodopsin itself is certainly an oversimplification. The strongest evidence in favour of the "calcium transmitter hypothesis" is the large calcium efflux from rods in a retina. However as long as the source of the calcium efflux inside the rod cells is unknown conclusions about the role of this calcium efflux are premature. Unfortunately, measurements of intracellular calcium, such as those by Brown and coworkers (93,94) in their pioneering work on photoreceptors in the ventral eye of Limulus, have not yet been feasible in vertebrates.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

On the role of sulfolipids in mammalian metabolism

Summary Sulfolipids of mammalian origin include sulfosphingolipids, sulfoglycerolipids and steroid sulfates. Sulfosphingolipids (sulfogalactosylceramide) may be involved in sodium transport, interaction of opiates with their receptors, activation of oxygen radical generating system, and blood coagulation Factor XII. Sulfoglycerolipids and steroid sulfates may be involved in spermatogenesis and sperm capacitation.     

Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK _m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

Arginase from rat fibrosarcoma. Its possible role in proline,glutamate and polyamine metabolism

The presence of arginase in rat fibrosarcoma not synthesizing urea, suggested that this enzyme may have additional functions. Ornithine carbamoyl transferase, a key enzyme of the urea cycle was absent in this tissue, when compared to normal tissues, lower amount of ornithine was found in the fibrosarcoma, but this tumour contained a higher level of proline. The radioactivity present in L-[U-¹⁴C] arginine was incorporated into putrescine, spermidine, spermine, proline glutamate and glutamine suggesting that arginine was a possible precursor and that arginase may have a role in the synthesis of these metabolites.     

Abscisic acid mutants affect polyamine metabolism

ABSTRACT

Flacca, tomato mutant in which the last step of ABA biosynthesis is blocked, displays changes both in polyamine content (especially the ratio between free and conjugated forms) and in ornithine-, arginine- and S- adenosylmethionine decarboxylase activities. Arginine decarboxylase activity is higher compared to that of ornithine decarboxylase especially in older plants (seven internode stage), being on line with its regulatory role. However this activity is not sufficient to justify the polyamine content, in particular of conjugated forms (disappearance of TCA-insoluble conjugates and dramatic decrease of TCA-soluble ones). These results are in favour of a multifunctional competition of many responses to hormone action.     

Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. Characterization of gene deletion mutants

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and Deltaarg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The Deltaarg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the Deltaarg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the Deltaarg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.     

Isoproterenol-induced responses in the regional polyamine metabolism of the rat heart

Rats were treated with a single dose of isoproterenol (25 mg/kg s.c.) and the levels of polyamines determined in various parts (right ventricle, basis, medial part and apex of the left ventricle) of the heart 24, 48 and 72 hours after the injection. The isoproterenol treatment produced marked alterations in the concentrations of cardiac polyamines. The most apparent changes were seen in the apex and medial part of the left ventricle where spermidine concentration exhibited a biphasic response with peaks at 24 and 72 hours. In the basis of the heart the spermidine concentration was significantly elevated only at 24 hours. In the right ventricle the spermidine level was significantly higher than control at 72 hours. Spermidine/spermine ratio was augmented in all cardiac tissues examined over the 72-hour period. Results appear to show that the isoproterenol-induced alterations in cardiac polyamine metabolism were not uniformly distributed in the various regions of the heart.     

Modeling the role of incisures in vertebrate phototransduction

Phototransduction is mediated by a G-protein-coupled receptor-mediated cascade, activated by light and localized to rod outer segment (ROS) disk membranes, which, in turn, drives a diffusion process of the second messengers cGMP and Ca2+ in the ROS cytosol. This process is hindered by disks-which, however, bear physical cracks, known as incisures, believed to favor the longitudinal diffusion of cGMP and Ca2+. This article is aimed at highlighting the biophysical functional role and significance of incisures, and their effect on the local and global response of the photocurrent. Previous work on this topic regarded the ROS as well stirred in the radial variables, lumped the diffusion mechanism on the longitudinal axis of the ROS, and replaced the cytosolic diffusion coefficients by effective ones, accounting for incisures through their total patent area only. The fully spatially resolved model recently published by our group is a natural tool to take into account other significant details of incisures, including their geometry and distribution. Using mathematical theories of homogenization and concentrated capacity, it is shown here that the complex diffusion process undergone by the second messengers cGMP and Ca2+ in the ROS bearing incisures can be modeled by a family of two-dimensional diffusion processes on the ROS cross sections, glued together by other two-dimensional diffusion processes, accounting for diffusion in the ROS outer shell and in the bladelike regions comprised by the stack of incisures. Based on this mathematical model, a code has been written, capable of incorporating an arbitrary number of incisures and activation sites, with any given arbitrary distribution within the ROS. The code is aimed at being an operational tool to perform numerical experiments of phototransduction, in rods with incisures of different geometry and structure, under a wide spectrum of operating conditions. The simulation results show that incisures have a dual biophysical function. On the one hand, since incisures line up from disk to disk, they create vertical cytoplasmic channels crossing the disks, thus facilitating diffusion of second messengers; on the other hand, at least in those species bearing multiple incisures, they divide the disks into lobes like the petals of a flower, thus confining the diffusion of activated phosphodiesterase and localizing the photon response. Accordingly, not only the total area of incisures, but their geometrical shape and distribution as well, significantly influence the global photoresponse.     

Inhibitors of polyamine metabolism: Review article

Summary. The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases.The early development of polyamine biosynthetic single enzyme inhibitors such as -difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer.The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N¹-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.