Synthetic Polyamines Research Articles

Molecular Requirements for Polyamines Binding to the Antispermine Monoclonal Antibody Spm8-2

The monoclonal antispermine antibody Spm8-2 was obtained by immunizing mice with a thyroglobulin-spermine conjugate. The molecular requirements for polyamines binding to this antibody were investigated by ELISA binding and inhibition tests, using a variety of natural polyamines and synthetic polyamine analogs. Four major structural determinants are important for the binding of polyamines by the antibody: (1) terminal amino groups: N-alkylation of both terminal amino groups of the polyamines leads to an important drop in the affinity for the antibody; (2) number of methylene groups spacing the amino groups: the four carbon chains appear to present the optimum length since the antibody binds polyamines with repeats of the aminobutyl moiety more actively than their homologues with shorter or longer carbon chains; (3) number of amino groups: the affinity of Spm8-2 for free homologous polyamines varied in the following order: pentamines > tetramines > triamines > diamines, showing the importance of the number of positive charges of the polyamines in the antibody-antigen reaction; the importance of charges is further emphasized by the dependence of antibody binding on the ionic strength of the medium; (4) N-acylation of one terminal amino group: the antibody binds more actively N1-acetylspermidine than spermidine or spermine. The binding properties of Spm8-2 suggest the presence of two recognition sequences, one selective for N-acylaminopropyl moieties, the second for the aminobutyl moiety.

Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex

Polyamines can modulate activation of N-methyl- d-aspartate (NMDA) receptors by binding to a specific polyamine site associated with a NMDA receptor macrocomplex. Polyamines induce histamine release from mast cells, although the mechanism had not been defined. We have examined whether spermine, a natural polyamine, and compound 48 80 , regarded as a synthetic polyamine, activate mast cells by a polyamine site associated with a NMDA receptor macrocomplex. Spermine induced secretion of histamine from rat peritoneal mast cells and rat brain mast cells in a concentration-dependent manner. Rat peritoneal mast cells were used as a model system to explore the effects of NMDA antagonists on polyamine-induced histamine release. Ifenprodil, MK801 and arcaine inhibited histamine secretion from mast cells exposed to polyamines; the percentage inhibition was greater against spermine than compound 48 80 . These data support the proposal that spermine (and possibly compound 48 80 ) induce histamine release from mast cells by interacting with a specific polyamine site on a NMDA receptor complex.

Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells.

Deregulation of polyamine transport in L1210 cells overexpressing ornithine decarboxylase leads to a lethal accumulation of spermidine. We now provide evidence that over-accumulation of natural and synthetic polyamines, but not putrescine, rapidly induces apoptosis, as shown by hypercondensation of peripheral chromatin and internucleosomal cleavage, followed by nuclear fragmentation. Polyamine oxidation is not responsible for the apoptosis observed. Thus, abnormally high polyamine pools could be an important physiological trigger of apoptosis.

Inactivation of human immunodeficiency virus type 1 by the amine oxidase-peroxidase system.

Human immunodeficiency virus type 1 (HIV-1) is rapidly inactivated by exposure to a naturally occurring antimicrobial system consisting of peroxidase, H2O2, and a halide. Among the potential sources of H2O2 is the amine oxidase system in which mono-, di-, and polyamines are oxidatively deaminated with the formation of H2O2. The polyamine spermine is present at exceptionally high concentrations in semen. We report here that spermine, spermidine, and, to a lesser degree, the synthetic polyamine 15-deoxyspergualin are viricidal to HIV-1 when combined with amine oxidase and myeloperoxidase. Antiviral activity required each component of the spermine-amine oxidase-peroxidase system and was inhibited by azide (a peroxidase inhibitor) and by catalase but not by superoxide dismutase. Heat treatment of catalase largely abolished its inhibitory effect. These findings implicate H2O2 formed by the amine oxidase system in the antiviral effect and raise the possibility that the polyamine-amine oxidase-peroxidase system influences the survival of HIV-1 in semen and in the vaginal canal.

Diethylhomospermine, a synthetic polyamine analog, reduces the extent of acetic acid-induced colonic mucosal damage in rats: T.A. Broome, R.J. Bergeron, G.Y. Lauwers, and C.A.Sninsky, Depts of Physiol Science, Medicine and Medicinal Chemistry, Un. of Florida and VAMC, Gainesville, Florida

Diethylhomospermine, a synthetic polyamine analog, reduces the extent of acetic acid-induced colonic mucosal damage in rats: T.A. Broome, R.J. Bergeron, G.Y. Lauwers, and C.A.Sninsky, Depts of Physiol Science, Medicine and Medicinal Chemistry, Un. of Florida and VAMC, Gainesville, Florida

Inhibition of Mitochondrial Permeability Transition by Polyamines and Magnesium: Importance of the Number and Distribution of Electric Charges

The protective effects of Mg 2+ and various natural and synthetic polyamines on the permeability transition of isolated rat liver mitochondria have been compared. The permeability transition was induced by incubating the mitochondria in a sucrose medium at pH 7.4 in the presence of 100 μM Ca 2+ and 1 mM phosphate and was monitored via the release of endogenous Mg 2+, sucrose permeation, mitochondria swelling and the fall of transmembrane potential. By all of these parameters (only the traces of ΔΨ have been reported) spermine fully inhibited the transition at 25 μM concentration, spermidine and caldine at 250 μM and Mg 2+ at 500 μM concentration. Both putrescine and dien exhibited only a partial protection even at 2.5 mM concentration. The protective action resulted strictly dependent on the number of the positive charges of each cation. In the case of polyamines this number is also determined by the nature of the methylene carbon chains of each compound.

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.

Histamine release from mast cells by polyamines: an NMDA receptor-mediated event?

In recent years it has become apparent that the role of the mast cell is not confined to immediate hypersensitivity reactions and that the cell may participate in a range of nonIgE mediated processes. The natural polyamines induce histamine release from rat peritoneal mast cells (RPMC) in a concentrationand energy-dependent process with a rank order of potency of spermine > spermidine > putrescine [ l l ; compound 48/80, a synthetic polyamine, is regarded as a classic mast cell secretagogue 111. However, the mechanism by which these agents trigger secretion in this system remains to be elucidated. It has been variously suggested that they interact with mast cells via direct stimulation of G-proteins 111 or with ill-defined polyamine 'receptors' [21. We have explored the possibility that spermine triggers histamine release from RPMC via interaction with a polyamine site associated with the NMDA receptor complex.

Polyamines and polyamine amides from wasps and spiders.

Introduction One of the aims of our research programme is to determine the molecular architecture of the ion channel buried deep within certain subtypes of glutamic acid-gated receptor (GluR), i.e. ionotropic not metabotropic GluR. This will require an interdisciplinary approach, but selective pharmacological tools are now available with which to begin to classify GluR subtypes. Such tools include polyamine amides, synthetic analogues of certain arthropod toxins. Another aspect of this research is the application of these channel blockers to related signalling systems, e.g. voltage-sensitive Caz+ channels and nicotinic acetylcholine receptors (nAChRs), themselves ligand-gated ion channels (LGICs). Therefore, a practical procedure, to achieve these aims, is the design and development of potent, selective channel blockers, followed by their affinity (radioand/or photo-) labelling for subsequent more detailed studies of the threedimensional array of the ion channels. At the commencement of these studies, polyamine amides were unknown as a class of natural products. We now recognize them as including the most potent, selective non-competitive antagonists of certain LGIC receptors. They are amenable to synthetic manipulation, but their high basicity makes them capricious to work with. Polyamines scavenge carbonic acid from the air, they are preferentially soluble in aqueous rather than organic media, and their preparation often requires a number of protecting and deprotecting steps during the synthetic sequence. Nevertheless, these arthropod venom toxins are potentially powerful tools in basic neuroscience. We have also considered the sites and modes of action of the natural and synthetic polyamine amides and modes of action of the natural and synthetic polyamine amides as a function of their pharmacological selectivity. If the

Regulation of the Phosphoinositide Cascade by Polyamines in Brain

The endogenous polyamines spermidine and spermine enhanced guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S)-stimulated phosphoinositide turnover with EC50 values of 100 +/- 30 and 50 +/- 15 microM, respectively, whereas the synthetic polyamines N,N'-bis(3-aminopropyl)-1,3-propanediamine and -ethylenediamine inhibited GTP-gamma-S-stimulated phosphoinositide turnover, with maximal inhibition at 1 mM. Kinetic analysis of GTP-gamma-S-stimulated phosphoinositide turnover in the absence and presence of spermidine showed that the Km for GTP-gamma-S was not changed (1,303 +/- 270 and 1,069 +/- 214 nM, respectively), whereas the Vmax was increased by 206% (1,566 +/- 141 and 4,792 +/- 84 cpm, respectively), indicating that spermidine and GTP-gamma-S acted at different sites. Spermidine also enhanced Ca(2+)-stimulated phosphoinositide turnover in the absence of GTP-gamma-S by decreasing the Ca2+ requirement of the phosphoinositide-specific phospholipase C. Arcaine and agmatine, polyamine antagonists at the NMDA receptor complex, did not block the effects of spermidine on GTP-gamma-S- and Ca(2+)-induced phosphoinositide turnover, suggesting that the spermidine effects are not mediated through these specific polyamine sites. Furthermore, spermidine increased the level of [3H]phosphatidylinositol 4-phosphate (EC50 = 120 +/- 10 microM), without affecting significantly the levels of [3H]-phosphatidylinositol and [3H]phosphatidylinositol 4,5-bis-phosphate. Collectively these data indicate that the enhanced phosphoinositide turnover induced by spermidine in the presence of GTP-gamma-S or Ca2+ is mediated through multiple levels of the phosphoinositide turnover cascade.

Diethylhomospermine, a synthetic polyamine analog, prevents CRF-induced acceleration of colonic transit in rats

Diethylhomospermine, a synthetic polyamine analog, prevents CRF-induced acceleration of colonic transit in rats

Influence of polyamines on growth and metabolism ofDunaliella primolecta

Addition of natural polyamines (putrescine, spermidine or spermine) to the culture medium stimulates growth of Dunaliella primolecta and results in an increased photosynthesis (+200%), chl a content (+300%) and ATP content (+182%). The results are compared with those induced by synthetic polyamines, such as N,N'-bis (3-aminopropyl) diaminoethane (BAPDE) or 1,4,8,11,tetra-azacyclotetradecane (CYCLAM). BAPDE and CYCLAM increase photosynthesis, ATP level and chl a content to the same extent, but have a negative effect on the growth of the alga. The synthetic polyamines increase oxygen uptake, whereas the natural polyamines have no effect. These results are discussed in relation to the hypothesis of polyamines acting as growth regulators or as nitrogen source for the alga.

IgG from amyotrophic lateral sclerosis patients increases current through P-type calcium channels in mammalian cerebellar Purkinje cells and in isolated channel protein in lipid bilayer.

The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, omega-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.

Potentiation by polyamines of an interaction of noncompetitive antagonists at the N-methyl-d-aspartate receptor ionophore complex with phosphatidylserine

The addition of phosphatidylserine induced a significant interaction with radioligands widely used for labeling an ion channel associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, such as [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) and [3H]N-[1-(2-thienyl)cyclohexyl]piperidine, in the absence of brain synaptic membranes irrespective of the addition of either L-glutamic acid or glycine when determined in the presence of the polyamine spermidine. The interaction with [3H]MK-801 increased in proportion to increasing concentrations of phosphatidylserine added at concentrations from 0.01 to 0.5 mg/ml. Similar interaction with [3H]MK-801 occurred for phosphatidylinositol to a lesser extent than for phosphatidylserine but not for either phosphatidylethanolamine or phosphatidylcholine at the similar concentration range. The interaction between phosphatidylserine and [3H]MK-801 was dependent partially on incubation temperature and on incubation time with reversible and saturable profiles. Among various natural and synthetic polyamines, both spermine and bis-(3-aminopropyl)amine in addition to spermidine were effective in markedly potentiating the interaction in a concentration-dependent manner at concentrations from 1 microM to 10 mM. However, the potentiation by spermidine was insensitive to antagonism by the respective antagonists for the NMDA and glycine domains at 0.1 mM. Moreover, the interaction between phosphatidylserine and [3H]MK-801 was sensitive to inhibition by several compounds with an antagonistic activity at calmodulin. The addition of phosphatidylserine completely concealed the potentiation by either glutamate alone or both glutamate and glycine of the binding of [3H]MK-801 in brain synaptic membranes with concomitant enhancement of the ability of spermidine to potentiate the binding, while phosphatidylserine failed to affect the affinities of the respective ligands for the NMDA and glycine domains. These results suggest that the acidic phospholipid phosphatidylserine may at least in part play crucial roles in mechanisms underlying the potentiation by polyamines of the binding of [3H]MK-801 to the open NMDA channel in rat brain.

Identification and characterization of specific binding sites of [ 3H]spermidine in synaptic membranes of rat brain

Synaptic membranes of rat brain contained specific binding sites of [ 3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [ 3H]SPD binding and to potentiate [ 3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N- methyl- d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [ 3H]SPD binding sites. Furthermore, [ 3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.

Modulation of the NMDA receptor by polyamines

Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. This site appears to be distinct from previously described binding sites for glutamate, glycine, Mg ++, Zn ++, and open-channel blockers such as MK-801. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neurons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of [ 3H]MK-801 and on NMDA-elicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to effect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.

Natural and Synthetic Polyamine Derivatives as Antagonists of Glutamate Receptors

Natural and Synthetic Polyamine Derivatives as Antagonists of Glutamate Receptors

G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides

The neuropeptide substance P, the venom peptide mastoparan and the synthetic polyamine compound 48/80 activate rat peritoneal mast cells, leading to rapid histamine release by exocytosis. Although these effects are inhibited by pertussis toxin and involve a transient increase in IP3, no selective membrane receptors have been identified. However, it has recently been shown that these compounds activate G proteins in vitro. Here Yves Landry and colleagues discuss the proposal that direct activation of G protein is the physiological mechanism of action of substance P on rat peritoneal mast cells, this mechanism being mimicked by mastoparan and 48/80, and possibly by other cationic amphiphilic peptides such as kinins. These compounds might be of help m defining the interaction between membrane receptors and G proteins.

Natural and synthetic polyamine derivatives as antagonists of glutamate receptors: an emerging structure/ activity profile

Natural and synthetic polyamine derivatives as antagonists of glutamate receptors: an emerging structure/ activity profile

Philanthotoxins. A mass spectrometric investigation

A method employing liquid secondary-ion mass spectrometry (SIMS) in conjunction with metastable-ion measurements (linked scanning at constant B/E) to obtain sequence-specific information for three synthetic polyamine isomers was developed. The normal liquid SIMS spectra gave molecular weight information, but important sequence ions were of low intensity or obscured by the background. The metastable-ion spectra contained important fragment ions in particular due to cleavage along the polyamine chain. One of the three synthetic isomers was identical with a toxin present in the venom of the digger wasp. In conjunction with nuclear magnetic resonance spectroscopic studies, this should be a powerful method for the structural characterization of other closely related toxins present in the venom of this wasp.