Pheromonal influences

The mechanism of uptake of sparfloxacin, a new quinolone, by intestinal brush-border membrane vesicles was investigated to clarify whether there is a common transport process for new quinolones mediated by the diffusion potential across the intestinal membrane bilayer. Sparfloxacin was taken up pH-dependently by rat intestinal brush-border membrane vesicles, behaviour analogous to that of organic cations including enoxacin and ciprofloxacin. Transient overshooting uptake of this quinolone was observed in the presence of an outward H+ gradient. Momentary dissipation of the H+ gradient by addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone did not affect the uptake of sparfloxacin, and a marked but incomplete reduction in the H+-sensitive overshooting uptake of sparfloxacin was apparent in the voltage-clamped brush-border membrane vesicles. Furthermore, a valinomycin-induced K+-diffusion potential (interior negative) and an inward C1--diffusion potential stimulated the initial uptake of sparfloxacin at pH 5.5. Sparfloxacin uptake was inhibited by tetracaine and imipramine. The inhibitory effect of these cations correlated well with changes in membrane surface charges induced by the presence of tetracaine or imipramine. These results indicate that sparfloxacin transport across the brush-border membrane depends upon the inside-negative ionic diffusion potential, that the H+- or K+-diffusion-potential-dependent uptake of sparfloxacin by intestinal brush-border membrane vesicles is affected by the membrane surface potential and that inhibition of sparfloxacin uptake originates from changes in the membrane surface potential caused by the organic cations.     

Effect of membrane surface potential on the uptake and the inhibition of cationic compounds in rat intestinal brush-border membrane vesicles and liposomes

The effect of membrane surface potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was investigated. In the presence of an inside-negative K(+)-diffusion potential, the manner of initial uptake of tryptamine appeared to be pH-dependent and the uptake in the acidic medium was lower than that in the neutral medium. Changes in surface potential of brush-border membrane vesicles were monitored using 8-anilino-1-naphthalenesulfonic acid (ANS) and the results suggested that the membrane surface potential (negative charge on the membrane surface) decreased in the acidic medium. A good correlation was observed between the K(+)-diffusion potential-dependent uptake of tryptamine and membrane surface potential monitored by ANS at various pH levels. The uptake of tryptamine by liposomes (large unilamellar vesicles), which contained various amounts of dipalmitoylphosphatidylserine (DPPS), was also examined. The uptake of tryptamine decreased with a decrease of DPPS content in the liposomes, and was correlated with the membrane surface potential monitored by ANS. Moreover, the effect of organic cations on the uptake of tryptamine by intestinal brush-border membrane vesicles was examined. The uptake of tryptamine was inhibited by tetracaine and imipramine. The inhibitory effect of these cations was well correlated with changes in the membrane surface potential in the presence of tetracaine or imipramine. These results suggest that the K(+)-diffusion potential-dependent uptake of tryptamine by intestinal brush-border membrane vesicles is affected by membrane surface potential, and the inhibition of tryptamine uptake originates in changes in the membrane surface potential caused by the organic cations.     

The involvement of polyamines in the activation of vitamin D receptor from porcine intestinal mucosa

In the intracellular process of the action of calcitriol, vitamin D receptor is thought to undergo some kind of physiochemical change, called activation, before the receptor binds to the vitamin D response element of the gene. In this paper, the effects of polyamines and their analogues on the sedimentation properties of vitamin D receptor prepared from porcine intestinal mucosa, and on DNA binding activity of the receptor, were studied. In sucrose density gradient analysis, polyamines decreased the sedimentation coefficient of vitamin D receptor in a dose-dependent fashion. Polyamines increased DNA binding activity of vitamin D receptor dose-dependently. These findings show that polyamines can activate vitamin D receptor in vitro. Among naturally existing polyamines, spermidine and spermine, but not putrescine, were effective within their physiological intracellular concentrations, suggesting that both spermidine and spermine can activate vitamin D receptor in vivo as well. Sucrose density gradient analysis using various kinds of polyamine analogues having various numbers of cations showed that the number of cation of polyamines is important for the efficiency to change the sedimentation coefficient of vitamin D receptor, and that the distance between two cationic charges does not play an important role.     

Female urethral syndrome. A female prostatitis?

N-Acetyltransferase, which is suggested to be responsible for the production of N1-acetylspermidine in Leishmania amazonensis and to be involved in the process of inactivation and degradation of excessive polyamines, was partially purified and characterized. Among the substrates tested, sym-norspermidine, sym-norspermine, and 1,3-diaminopropane had the highest reaction rates, but the naturally occurring polyamines spermine and spermidine were also acetylated at considerable rates, whereas putrescine was a poor substrate. The Michaelis constants (Km values) for spermine and spermidine were 0.66 and 3.3 mM, respectively. The Km value for acetylcoenzyme A (acetyl-CoA) was determined to be 34 microM. CoA inhibited the reaction in a competitive manner; the inhibition constant was 5 microM. The enzyme showed an apparent relative molecular mass of 35,000.     

Characterization of a diamine exporter in Chinese hamster ovary cells and identification of specific polyamine substrates

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.     

Modulation of putrescine transport in rat intestinal brush-border membrane vesicles by fasting and refeeding

Fasting and refeeding dramatically alter small intestinal mucosal growth which is greatly dependent on polyamine biosynthesis and transport. The aim of this study was therefore to examine the uptake of the diamine putrescine by brush-border membrane vesicles from the small intestine of rats fasted for 3 days or refed a standard diet after a PERIOD OF FASTING. WHILE THE MICHAELIS-MENTEN CONSTANT KM WAS essentially unaltered, the maximum velocity (Vmax) for putrescine uptake was 1.85-fold higher in fasted animals than in ad libitum-fed controls. Refeeding fasted rats for 24 h caused a 31% decrease in the Vmax value that, however, remained 1.27-fold higher than in control RATS, WHILE THE KM VALUE WAS STILL UNCHANGED. FASTING RATS OR refeeding rats after a period of fasting caused only a 13 or 17% increase, respectively, in the value of the constant for the nonsaturable component (P) of putrescine transport relative to the corresponding control condition. Our study also confirms that both the mucosal polyamine biosynthesis and intestinal content are altered by fasting. We suggest that an increased uptake activity may have a conservative role by preventing a substantial loss of tissue polyamines during fasting.     

Intestinal absorption of peptides by coupling to bile acids

Poor intestinal absorption of peptides greatly limits their use as drugs for the treatment of chronic diseases. Since bile acids are efficiently absorbed by an active, Na(+)-dependent transport system in the ileum of mammals, model peptides of different chain length were attached to the 3-position of modified 3 beta-(omega-amino-alkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid. These peptide-bile acid conjugates inhibited Na(+)-dependent [3H]taurocholate uptake into brush-border membrane vesicles isolated from rabbit ileum in a concentration-dependent manner. Furthermore, photoaffinity labeling of the bile acid-binding proteins of M(r) 93,000 and 14,000, identified as the protein components of the ileal Na(+)-dependent bile acid transport system in rabbit ileum (Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Müller, G., Tripier, D., and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046) by the photoreactive taurocholate analogue, (3,3-azo-7 alpha, 12 alpha-dihydroxy-5 beta [7 beta, -12 beta-3H]cholan-24-oyl)-2-aminoethanesulfonic acid, was inhibited by the peptide-bile acid conjugates. In contrast, the parent peptides and amino acids neither had a significant effect on [3H]taurocholate uptake by ileal brush-border membrane vesicles nor on photoaffinity labeling of the ileal bile acid-binding membrane proteins. The inhibitory effect of peptide-bile acid conjugates on [3H]taurocholate transport and photoaffinity labeling of the bile acid-binding proteins in rabbit ileal vesicles decreased with increasing chain length of the attached peptide radical. By in vivo ileum perfusion in anesthetized rats an intestinal absorption of the bile acid conjugate S3744 of the fluorescent oxaprolylpeptide 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-Opr-Gly (S1037) and secretion of the intact compound into bile could be demonstrated, whereas the parent peptide S1037 or its t-butylester S4404 were not absorbed. The intestinal absorption of S3744 showed a similar temperature dependence as [3H]taurocholate absorption and was inhibited by the presence of taurocholate indicating a carrier-mediated uptake of S3744 via the ileal bile acid transporter. In conclusion, these results indicate that oligopeptides can be made enterally absorable by coupling to modified bile acid molecules making use of the specific intestinal absorption pathway for bile acids. This finding may be of great importance for the design and development of orally active peptide drugs.     

Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli

A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl beta-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to N1-acetylspermidine, much of which was excreted. Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine. These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more sensitive to the growth inhibitory action of the antitumor agent N1,N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5 alpha cells containing pINSAT2. The purified enzyme had a similar specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.     

Polyamine metabolism in cystic fibrosis

Increased blood levels of spermidine and an increased spermidine/spermine ratio is documented in 31 patients with cystic fibrosis (CF). Some 29 percent of CF patients overlap with the control group. An abnormality of urinary free polyamines or their metabolic derivatives related to electrolyte transport, serum ciliostatic factors, and the autonomic nervous system are elaborated upon.     

Proton-cotransport of pravastatin across intestinal brush-border membrane

PURPOSE: The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. METHODS: The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. RESULTS: The initial uptake of [14C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pHin/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [14C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [14C]pravastatin in the presence of a potassium gradient ([K+]in/[K+]out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [14C]pravastatin. In the presence of a proton gradient (pHin/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent Kt of 15.2 +/- 3.2 mM and Jmax of 10.6 +/- 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tricarboxylic acids, or acidic amino acid. CONCLUSION: It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Immunocytochemical localization of polyamines in the tiger salamander retina

The polyamines spermine and spermidine are present in neural tissue, but their functions there are not well understood. Recent work suggests that the NMDA subtype of glutamate receptors, other glutamate receptor subtypes, and certain K(+)-channels, are neural targets for polyamines. To better understand the neuron-specific roles of polyamines, we have developed antibodies that interact with spermine and spermidine in aldehyde-fixed tissue and used these antibodies in immunocytochemical studies to determine the cellular localization of these polyamines in the tiger salamander retina. The affinity-purified, polyclonal antibodies were highly specific for spermine and spermidine, exhibiting < 1% cross reactivity with putrescine, and virtually no cross-reactivity with GABA, arginine, lysine, or glutaraldehyde. Polyamine labeling was most abundant in cells in the inner half of the inner nuclear layer and in the ganglion cell layer. Some cells in the outer half of the inner nuclear layer are labeled, and there was some labeling in both synaptic layers. Double-labeling experiments indicated (1) all GABAergic amacrine cells were polyamine-positive; and (2) all ganglion cells (identified by back-filling after microinjections of rhodamine in the optic nerve) were polyamine-positive. These results are consistent with a role for polyamines as modulators of NMDA receptor function and channel function in the inner retina.     

Type I hyperprolinemia: a study of the intestinal absorption of proline, hydroxyproline, and glycine

Intestinal absorption of proline, hydroxyproline, and glycine was interpreted by investigation of a type I hyperprolinemia patient and six control subjects. Intestinal perfusion was performed. When proline (Pro), hydroxyproline (OH-Pro), and glycine (Gly) were infused together, an increase in proline concentration did not alter aminoacid uptake in the control subjects; however, in the hyperprolinemia patient, uptake of aminoacids became negliglible (Pro, 17--6 muM/min; OH-Pro, 15--0.3 muM/min; and Gly, 13.5--0 muM/min). When each aminoacid was infused alone at increasing concentrations aminoacid uptake increased in controls; in the hyperprolinemic patient, intestinal absorption was less for glycine and hydroxyproline but aminoacid uptake increased with substrate concentration; however, for proline, the uptake remained constant (1l.5--17 muM/min/20 cm of intestinal test segment) (Table 1). When hydroxyproline was infused with an increased concentration of proline in the hyperprolinemic patient, hydroxyproline uptake first increased (9.8--14.3 muM/min/20 cm) then decreased to its basal value, whereas, in the control subjects, uptake increased without decreasing subsequently.     

Uptake of doxifluridine by rabbit small intestinal brush-border membrane vesicles

Characteristics of the transport of doxifluridine (5'-DFUR) in rabbit small intestinal brush-border membrane vesicles were examined. 5'-DFUR was rapidly incorporated and a linear relation was found between the concentration of 5'-DFUR and its uptake rate in the concentration range tested (0.1-10 mM). The uptake rate increased at an acidic pH, at which 5'-DFUR is present mostly in an undissociated form. However, the uptake rate was not changed either by the extravesicular Na+ concentration or by the inside negative membrane potential. Nucleosides, uridine and adenosine only slightly inhibited the uptake, even at a fifty-fold concentration, and nucleobases, uracil and thymine had no effect. These results suggest the possibility of the major involvement of passive diffusion of the undissocated form of 5'-DFUR in the uptake of the drug through the brush-border membrane at a relatively high concentration.     

Ornithine decarboxylase inactivation by putrescine: involvement of lipid peroxidation

Effect of polyamines on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced reduction of lipid peroxidation was studied. Putrescine protected this lowering of lipid peroxidation in a concentration-dependent manner, but spermidine or spermine could not do so. Putrescine also inhibited the TPA-induced ornithine decarboxylase (ODC) activity and lowered the free sulfhydryl content of TPA exposed mouse skin. These observations indicate that putrescine inactivates ODC probably by lowering SH groups through lipid peroxidation.     

Intracellular distribution of polyamines in human lymphoblastoid cell line during phorbol ester-induced differentiation

Polyamines such as putrescine, spermidine and spermine play an important role in nucleic acid metabolism. These aliphatic amines display a key role in cell-induced transformation by carcinogen substances. In particular, one of these, the phorbol myristate acetate, provokes cell differentiation and gives an increase of ornithindecarboxylase activity; enzyme regulating the pathways of polyamines. In this study we analyse the trend of the polyamines at cytoplasmic and nuclear level during phorbol treatment. Our results show a correlation between nuclear and cytoplasmic spermine, 3H-Thymidine, 3H-Leucine incorporation and cell cycle phases. These data remark that the polyamines are differently distributed into the cell during the phorbol myristate acetate-mediated differentiation process and that the spermine is down-regulated for to supply the increased protein biosynthesis.     

Practical recommendations and new therapies for Wilson's disease

Wilson's disease is an inherited disorder of copper accumulation. The basic defect is a failure of excretion of excess copper in the bile by the liver for loss in the stool. The accumulating copper causes damage primarily to the liver and the brain. Patients typically present in the second to the fourth decades of life with liver disease, a neurological disease of the movement disorder type, or a wide array of behavioural disturbances. Because the manifestations of Wilson's disease are so protean, and the disease masquerades so well as something else, recognition of the possibility of Wilson's disease is a major problem, leading to serious underdiagnosis of the disease. Excellent therapies exist for both the prophylaxis and treatment of Wilson's disease. The longer recognition and diagnosis are delayed, the greater the risk of permanent damage to liver and/or brain. The availability of effective therapy and the risks in delay or therapy make the earliest possible diagnosis critical. Once the disease comes under consideration, a series of diagnostic steps can be undertaken which almost always establish or rule out the diagnosis of Wilson's disease. These include urine copper, blood ceruloplasmin, slit lamp examination for Kayser-Fleischer rings, and liver biopsy with quantitative copper assay. Currently, there are 4 drugs being used as anticopper agents in Wilson's disease. These are zinc, which blocks intestinal absorption of copper, penicillamine and trientine, both of which are chelators that increase urinary excretion of copper, and tetrathiomolybdate which forms a tripartite complex with copper and protein, and can block copper absorption from the intestine, or render blood copper non-toxic. Zinc is clearly the treatment of choice, in our opinion, for maintenance therapy, for the treatment of the presymptomatic patient from the beginning and for the treatment of the pregnant patient, because of its complete efficacy and lack of toxicity. For the initial treatment of the patient presenting with mild liver failure, we empirically use a combination of trientine and zinc. Trientine gives a strong, fast, negative copper balance, and zinc induces hepatic metallothionein, which sequesters hepatic copper. For the initial treatment of patients presenting with neurological disease we use an experimental drug, tetrathiomolybdate, which provides rapid, safe control of copper. These latter patients are at great risk of serious permanent neurological worsening with penicillamine, and zinc is too slow-acting, in our judgment, to be optimal.     

Polyamine metabolism in Acanthamoeba culbertsoni

1,3-Diaminopropane has been identified as the major polyamine of Acanthamoeba culbertsoni. N-acetylputrescine and spermidine were present in appreciable amounts and putrescine as well as N-acetylspermidine were also detected, but spermine was absent. Changes in polyamine levels were observed during the growth of amoebae. Ornithine decarboxylase activity was detected in cell-free extracts but there was very low activity of arginine and lysine decarboxylases. A potent polyamine oxidase was demonstrated which preferentially acted on N8-acetyl-spermidine as the substrate while N1-acetylspermidine was a poor substrate; free polyamines did not serve as a good substrate for this enzyme. Active uptake of polyamines by the amoebae was also demonstrated.     

S-adenosylmethionine decarboxylase activity and utilization of exogenous putrescine are enhanced in colon cancer cells stimulated to grow by EGF

Polyamines spermidine and spermine and their precursor putrescine are necessary for cell growth. Polyamine content is high in rapidly growing malignant cells, due to enhanced putrescine synthesis by ornithine decarboxylase (ODC), and increased uptake. In contrast to other cells of the body, colon cancer cells are exposed to high putrescine concentrations from the lumen. AIMS: To investigate the utilization of luminal putrescine in colon cancer, we studied the effect of a potent mitogen, epidermal growth factor (EGF), on the activity of the enzyme responsible for putrescine conversion, S-adenosylmethionine decarboxylase (SAMDC), in Caco-2 cells. METHODS: Cell counts, ODC and SAMDC activities and intracellular polyamines were evaluated in the presence and absence of exogenous putrescine in concentrations resembling those normally present in the colonic lumen. RESULTS: ODC and SAMDC activity and putrescine uptake were strongly stimulated by EGF. Both synthesized and absorbed putrescine was rapidly converted to spermidine and spermine after EGF. Conversion pattern was identical in the cells stimulated with EGF only and EGF plus exogenous putrescine, indicating that, if stimulated to proliferate, colon cancer cells utilize the entire available putrescine pool. SAMDC inhibitor, methylglyoxal-bis-guanylhydrazone, induced growth arrest which was not reversed by exogenous putrescine, but only by high concentrations of spermidine. CONCLUSION: Enhanced proliferation in colon cancer cells is associated with increased SAMDC activity and rapid conversion of putrescine to spermidine and spermine. SAMDC might be a preferable target for therapeutic attempts to impair growth by reducing intracellular polyamine pools in colon cancer.