The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.
Blood Proteins ; Cholesterol* ; Genes, Reporter ; Introns ; Luciferases ; Oligonucleotides, Antisense ; Spermine*

PURPOSE: This study was designed to investigate the effects of polyamines on rabbit seminal vesicular contractility. MATERIALS AND METHODS: The polyamines; putrescine, spermidine and spermine, were added to deepithelized and precontracted seminal vesicle strips, with either 10 4M norepinephrine (NE), 10 4M acetylcholine (ACh) or 70mM KCl, in organ chambers to obtain cumulative concentration response curves. A whole cell mode patch clamp study was also performed to observe the effects of the polyamines on the L-type calcium channel activities. RESULTS: The polyamines elicited concentration-dependent relaxations of the precontracted strips with the NE, ACh and KCl. The spermine showed the most potent relaxation response. Both extracellular and intracellular application of the spermine decreased the L-type calcium channel currents. CONCLUSIONS: Spermine more potently inhibited the seminal vesicle contraction than putrescine or spermidine, which suggests the polyamines may play a role in maintaining the basal tonicity of seminal vesicle in a flaccid state. The spermine-induced relaxation response seems to be related with an inhibition of the L-type calcium channel activities.
Acetylcholine ; Calcium Channels, L-Type ; Norepinephrine ; Polyamines* ; Putrescine ; Relaxation* ; Seminal Vesicles* ; Spermidine ; Spermine

Interactions among dexamethasone, dehydroepiandrosterone (DHEA), lipopolysaccharide (LPS), and antimycin A on the glutamate uptake and the polyamine uptake were investigated in primary cultures of rat cerebral cortical astrocytes to examine the effects of dexamethasone and DHEA on the regulatory role of astrocytes in conditions of increased extracellular concentrations of glutamate or polyamines. 1. (3H)Glutamate uptake: LPS and antimycin A decreased Vmax, but both drugs had little effect on Km. Dexamethasone also decreased basal Vmax without any significant effect on Km. And dexamethasone further decreased the antimycin A-induced decrease of Vmax. DHEA did not affect the kinetics of basal glutamate uptake and the change by LPS or antimycin A. 2. (14C)Putrescine uptake: LPS increased Vmax, and antimycin A decreased Vmax. They showed little effect on Km. Dexamethasone decreased Vmax of basal uptake and further decreased the antimycin A-induced decrease of Vmax, and also decreased Vmax to less than control in LPS-treated astrocytes. DHEA did not affect Km and the change of Vmax by LPS or antimycin A. 3. (14C)Spermine uptake: Antimycin A decreased Vmax, and LPS might increase Vmax. Km was little affected by the drugs. Dexamethasone decreased basal Vmax and might further decrease the antimycin A-induced decrease of Vmax. And dexamethasone also decreased Vmax to less than control in LPS-treated astrocytes. DHEA might increase basal Vmax and Vmax of LPS-treated astrocytes. 4. Vmax of glutamate uptake by astrocytes was increased by putrescine (1000 muM & 2000 muM) and spermidine (200 muM, 500 muM & 2000 muM). Spermine, 200 muM (and 100 muM), also increased Vmax, but a higher dose of 2000 muM decreased Vmax. Km of glutamate uptake was not significantly changed by these polyamines, except that higher doses of spermine showed tendency to decrease Km of glutamate uptake. In astrocytes, dexamethasone inhibited the glutamate uptake and the polyamine uptake in normal or hypoxic conditions, and the polyamine uptake might be stimulated by LPS and DHEA. Polyamines could aid astrocytes to uptake glutamate.
Animals ; Antimycin A* ; Astrocytes* ; Dehydroepiandrosterone* ; Dexamethasone* ; Glutamic Acid* ; Kinetics ; Polyamines ; Putrescine ; Rats* ; Spermidine ; Spermine

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage- dependent calcium current (VDCCL). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (IBa), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC50=1.1+/-0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K+-induced contraction: Spermidine (5 mM) significantly reduced high K+(50 mM)-induced contraction to 37+/-4.7% of the control (p<0.05), and inhibitory effect of spermidine on IBa was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca2+-induced Ca2+release (CICR),` was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCCL and Ca2+releasing mechanism in guinea-pig stomach.
Acetylcholine ; Caffeine ; Calcium ; Calcium Channels ; Carbachol ; Contracts ; Muscle, Smooth ; Putrescine ; Relaxation ; Spermidine ; Spermine ; Stomach

Polyamines are non-specific marker of cellular proliferation in many malignant tumors, and it is also increase in certain benign conditions. We measured the urinary polyamines to investigate the possibility as a marker of abnormal prostate growth and the correlation with various clinical parameters. Urinary polyamine concentrations in 27 cases of symptomatic benign prostatic hyperplasia (BPH) were compared with those in 32 cases of age matched normal controls. Urinary concentration of polyamine profiles were quantitatively determined by Gas Chromatography/Nitrogen Phosphorus Detector and they were calculated by the correction of gram creatinine. The concentrations of N-acetyl putrescine, N-acetyl cadaverine, spermidine(spd), N1-acetyl spermidine, N8-acetyl spermidine, and spermine(spm) showed significant increase in BPH compared with normal control(all p<0.05). Level of serum prostate specific antigen(PSA) in BPH patients was negatively correlated with the concentration of urinary spermidine(p=0.049). And the ratio of spm/spd correlated with the level of prostate volume(p=0.046). No significant correlations was found between other clinical parameters such as age, level of hemoglobin or erythrocyte count with polyamine profiles concentration. These data suggested that urinary concentration of polyamines in BPH are elevated compared with those in normal control. Altered regulation of the biosynthesis and metabolism of spermidine and spermine may be involved in BPH.
Cadaverine ; Cell Proliferation ; Creatinine ; Erythrocyte Count ; Humans ; Metabolism ; Phosphorus ; Polyamines ; Prostate ; Prostate-Specific Antigen ; Prostatic Hyperplasia* ; Putrescine ; Spermidine ; Spermine

This work reports the properties of dextran-spermine polycation (DSP) as a gene vector and its gene transfection efficiency in vitro. Oxidized dextran was reacted by reductive amination with spermine to obtain DSP, the resulted polycation was then incubated with plasmid pEGFP to form polyplexes by electrostatic interactions. DSP formed stable polyplexes when the weight ratio (DSP/DNA) varied from 4 : 1 to 20 : 1. The particle size and zeta potential of polyplexes were in the range of 162.6 - 187.9 nm and increased from + 8.45 mV to + 39.6 mV, respectively. DSP could effectively protect condensed DNA from DNase I degradation, and it showed strong buffering capacity in a certain pH range. The highest yields of transfection efficiency were found to be as high as Lipofectamine 2000 when the polyplexes were transfected to SMMC-7721 and BHK-21 cells at the weight ratio of 8 : 1. This research indicates that dextran-spermine polycation is a highly active gene vector in vitro.
Animals ; Cell Survival ; Cricetinae ; DNA ; administration & dosage ; Dextrans ; administration & dosage ; Genetic Vectors ; Spermine ; administration & dosage ; Transfection ; methods

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.
Amino Acids ; Binding Sites ; Calcium ; Cytosol ; Glutamic Acid ; Myocytes, Smooth Muscle ; Permeability ; Polyamines ; Receptors, Muscarinic ; Spermine ; Transient Receptor Potential Channels

BACKGROUND AND OBJECTIVES: Effects of the three major endothelium-derived relaxing factors (EDRFs), namely nitric oxide (NO), prostacyclin (PGI2), and 11, 12-epoxyeicosatrienoic acid (EET) on the ATP-sensitive potassium channel (K ATP channel) activity were examined in isolated cardiac ventricular myocytes. MATERIALS AND METHODS: K ATP channel activities were measured in the enzymatically (collagenase) isolated single mouse ventricular myocytes using excised inside-out, cell-attached, and perforated whole-cell patch clamp techniques. RESULTS: In inside-out patches, NO donors, SNP and spermine NONOate, did not affect the K ATP channel activity. In the presence of both ATP and ADP in the bath solution, the NO donors attenuated the activity of the K ATP channel. In cell-attached patches, the NO donors potentiated pinacidil-induced K ATP channel activity. In perforated whole-cell patch configuration, the NO donors decreased the K ATP current induced by PCO 400, a K ATP channel opener. PGI2 did not affect the K ATP channel activity in excised insideout patch. However, in the pres-ence of ATP in the internal solution, PGI2 increased the channel activity in a dose-dependent manner. In cell-attached patches, PGI2 did not only affect the channel activity itself, but also the dinitrophenol-induced K ATP channel activity. 11, 12-EET had no effect on K ATP channel activities.CONCLUSION: These results indicate that some of the endothelium-derived relaxing factors (nitric oxide and prostacyclin) are involved in the regulation of ATP-sensitive potassium channel activities in mouse ventricular myocytes; and the regulation type was com-plicated, activation or inhibition, depending on the cellular environment.
Adenosine Diphosphate ; Adenosine Triphosphate ; Animals ; Baths ; Endothelium-Dependent Relaxing Factors* ; Epoprostenol ; Humans ; Mice ; Muscle Cells ; Myocytes, Cardiac* ; Nitric Oxide ; Patch-Clamp Techniques ; Potassium Channels* ; Potassium* ; Spermine ; Tissue Donors

BACKGROUND: In brain ischemia, increased arachidonic acid metabolism can play important roles in neuronal dam-age. Ibuprofen was reported to have a protective role against neuronal damage in focal brain ischemia and reperfusion. The present study was designed to investigate whether ibuprofen can inhibit the global ischemia-induced neuronal dam-age and changes of polyamine (PA) level which is known to related to the neuronal damage, breakdown of blood brain barrier, and brain edema. METHODS: Male Mongolian gerbils were used in this study. Transient global ischemia was induced by occlusion of bilateral common carotid arteries for 3 min with microclips. Ibuprofen was administered imme-diately after ischemia. The animals were sacrificed one day after ischemia for PA measurement and sacrificed 5 days after ischemia for histological evaluation. Histological examination was performed by counting surviving neuronal cells in one mm of CA1 area in dorsal hippocampus. RESULTS: Cerebral cortex and hippocampal putrescine(PU) levels in vehicle-treated ischemic group significantly increased comparing to sham-operated animals and the increase of PU was attenuated by ibuprofen administration (50 mg/kg). Hippocampal spermine level decreased significantly after ischemia. Hippocampal neuronal cell damage in CA1 area was markedly observed in vehicle-treated animals compared to sham operated animals. Ibuprofen administration at the dose of 50 mg/kg significantly inhibited hippocampal CA1 neuronal damage compared to vehicle-treated animals. CONCLUSIONS: Ibuprofen attenuates PA response following transient glob-al ischemia and may have putative neuroprotective effect against neuronal damage induced by global ischemia.
Animals ; Arachidonic Acid ; Blood-Brain Barrier ; Brain Edema ; Brain Ischemia ; Carotid Artery, Common ; Cerebral Cortex ; Gerbillinae* ; Hippocampus ; Humans ; Ibuprofen* ; Ischemia* ; Male ; Metabolism ; Neurons* ; Neuroprotective Agents ; Reperfusion ; Spermine

Glutamate Is the predominant excitatory neurotransmitter in the mammalian CNS. To elucidate the influence of glutamate on the noradrenergic neurotransmission in rat cortex, we examined the effects of agents that act in several steps of neurotransmission on [3H]norepinephrine ([3H])NE) release evoked by glutamate. Glutamate (1 mM) evoked significant release of [3H]NE from rat cortex slices in the absence of Mg2+in the incubation media. This effect was attenuated by cromakalime (10 nM) and lemakalime (10 nM), and the inhibitory effect of cromakalime was abolished by glipizide. Inhibitory effect of muscimol (30 uM) and baclofen (3 uM, 30 uM) was antagonized by biccuculine (3 uM), respectively. Nipecotic acid(10 uM), DABA(300 uM), and beta-alanine(100 uM) attenuated the glutamate-induced release of [3H]NE. Dihydrokinate (300 uM) PDC (100 nM) increased the glutamate-induced release of [3H]NE. Ifenprodile (10 nM) and arcaine (1 uN), blockers of polyamine site, attenuated the release of ("H)NE. The stimulatory effect of spermine was abolished by arcaine. CPA(100 nM) and CPCA(100 nM), EHNA(30 uN) and NBTI(1 uN) attenuated the release of ("H)NE. Verapamil(S uN), nitredipine(10 uN), u- conotoxin (100 nM) and flunarizine (5 uM) attenuated the release of (3H)NE. Dantrolene(30 uM), KT-362(3 uM), and ryanodine(10 nM), attenuated the glutamate-induced release of [3H]NE. Glycine (10 uM) increased the release of [3H]NE. DCQX (30 uN) attenuated the release of [3H]NE. These results suggest that glutamate-evoked release of norepinephrine can be modulated by GABAergic, adenosinergic neurotransmitters, and by various drugs which modulate ion channel activities in rat cortex.
Animals ; Baclofen ; Cerebral Cortex ; Conotoxins ; Cromakalim ; Flunarizine ; Glipizide ; Glutamic Acid ; Glycine ; Ion Channels ; Muscimol ; Neurotransmitter Agents ; Norepinephrine* ; Rats* ; Spermine ; Synaptic Transmission