Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria.

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.

Cytoplasmic inclusions resembling nucleoli in sympathetic neurons of adult rats.

A distinctive cytoplasmic inclusion consisting of a convoluted network of electron-opaque strands embedded in a less dense matrix was identified in the neurons, but not in the supporting cells, of rat sympathetic ganglia. This ball-like structure, designated "nematosome," measures approximately 0.9 micro and lacks a limiting membrane. Its strands (diameter = 400-600 A) appear to be made of an entanglement of tightly packed filaments and particles approximately 25-50 A thick. Cytochemical studies carried out with the light microscope suggest the presence of nonhistone proteins and some RNA. Usually only one such structure is present in a cell, and it appears to occur in most ganglion cells. Although they can be seen anywhere in the cell body, nematosomes are typically located in the perinuclear cytoplasm, where they are often associated with smooth-surfaced and coated vesicles. In fine structure and stainability, they bear a resemblance to the fibrous component of the nucleolus. Subsynaptic formations, which are a special feature of some of the synapses in sympathetic ganglia, appear similar to the threadlike elements in the nematosomes. The possibility that these three structures-nucleolus, nematosome, and subsynaptic formation-may be interrelated in origin and function is discussed.

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.

Transport of amino acids through the placenta and their role.

Amino acids are transported across the human placenta mediated by transporter proteins that differ in structure, mechanism and substrate specificity. Some of them are Na+-dependent systems, whereas others are Na+-independent. Among these there are transporters composed of a heavy chain, a glycoprotein, and a light chain. Moreover, they can be differently distributed in the two membranes forming the syncytiotrophoblast. The transport mechanisms involved and their regulation are only partially known. In the placenta itself, part of the amino acids is metabolized to form other compounds important for the fetus. This occurs for instance for arginine, which gives rise to polyamines and to NO. Interconversion occurs among few other amino acids Transport is altered in pregnancy complications, such as restricted fetal growth.

S-adenosylmethionine and its products.

S-adenosylmethionine is involved in many processes, mainly methylation, polyamine synthesis and radical-based catalysis. It is synthesised through the catalysis of differently regulated enzyme forms. When it is used, the compounds formed are reutilized in different ways: in case of methylation, its end product is homocysteine, which can be remethylated to methionine, give rise to cysteine in the so-called transsulphuration pathway, or be released; in the case of polyamine synthesis, the methylthioadenosine formed is cleaved and gives rise to compounds which can be reutilized; during radical-based catalysis, 5-deoxyadenosine is formed and this, too, is cleaved and reutilized.

Advanced glycation end-products (AGEs): involvement in aging and in neurodegenerative diseases.

Advanced glycation end-products (AGEs) are formed from the so-called Amadori products by rearrangement followed by other reactions giving rise to compounds bound irreversibly. The structure of some of them is shown and the mechanism of formation is described. Several AGE binding molecules (Receptors for AGE, RAGE) are known and it is thought that many of the effects caused by AGEs are mediated by RAGE. Some of these were shown to be toxic, and called TAGE. The mechanism of detoxification of glyoxal and methylglyoxal by the glyoxalase system is described and also the possibility to eliminate glycated proteins by deglycation enzymes. Compounds able to inhibit AGEs formation are also taken into consideration.

Modulation of ornithine aminotransferase activity by oxygen in rat hepatocyte cultures.

In hepatocytes in culture, ornithine aminotransferase activity remained higher when the cells were cultured at low oxygen tension (5%) than at high tension (21%), that is, it was higher in hepatovenous conditions. Northern blot analysis showed that the amount of the specific mRNA for the enzyme was also higher. Results of experiments performed in the presence of CoCl2, to replace the central Fe2+ in heme, or succinylacetone, to inhibit heme synthesis, support the view that a heme protein participates in the regulation of ornithine aminotransferase activity by oxygen. The oxygen sensor does not appear to act through phosphorylation by kinase C, as TPA has no significant effect on the process, but a phosphorylation dependent on cAMP might be involved.

Pharmacodynamic effects of cotinine in abstinent cigarette smokers.

The nicotine metabolite cotinine was administered to abstinent cigarette smokers to determine whether it has pharmacologic activity as assessed by various physiologic and subjective measurements. By means of a randomized, double-blind, placebo-controlled counterbalanced-order design, subjects received cotinine base (30 mg) intravenously after 48 hours of abstinence from cigarette smoking. Serum cotinine concentrations increased to levels commonly achieved during daily cigarette smoking, whereas no change in serum nicotine concentration was observed. Cotinine compared with placebo produced subjective differences in self-reported ratings of restlessness, anxiety and tension, insomnia, sedation, and pleasantness. Cotinine had minimal effects on cardiovascular measurements. These findings indicate that cotinine is behaviorally active in the setting of cigarette abstinence at blood concentrations similar to those commonly achieved through daily cigarette smoking.

Neutral red stains ganglia in the vagal motor pathway to ferret trachea without affecting ganglionic transmission.

To determine the effect of Neutral red (0.01%) on neural transmission through ganglia, we used an in vitro nerve-muscle preparation of ferret trachea. Before, during, and after incubating the trachea in Neutral red, we induced isometric muscle contractions first by activating preganglionic fibers with electrical stimulation of the vagus nerve, and then by activating postganglionic nerve fibers with electrical field stimulation. Incubation in Neutral red (0.01%) for 45 min at 38 degrees C reduced the responses to both pre- and postganglionic activation. When the control responses to pre- and postganglionic activation were matched. Neutral red depressed the 2 responses to the same degree, implying that the depression was confined to postganglionic structures. Washout of Neutral red from the medium restored the responses to both pre- and postganglionic activation. Histologic examination of all tissues proved that the ganglia were still stained after the washout procedure. We conclude that Neutral red (0.01%) depresses smooth muscle contractions evoked through neural pathways, and that this depression is reversible and confined to postganglionic structures, leaving ganglionic transmission intact.

Long chain diamines inhibit growth of C6 glioma cells according to their hydrophobicity. An in vitro and molecular modeling study.

A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.

Regulation of spermidine acetyltransferase activity by phosphorylation and dephosphorylation.

Spermidine acetyltransferase activity is more than 10-fold higher in the pancreas of a 20-hr-fasted than in that of a fed chicken. The preparation of the fed bird inactivates the other. The effect is due to a thermolabile component of microsomes, and is also obtained with alkaline phosphatase. The inactivated preparation partially recovers its activity through phosphorylation catalyzed by a cAMP-dependent protein kinase. The results presented strongly suggest that spermidine acetyltransferase activity is regulated by phosphorylation and dephosphorylation.

Carbamylphosphate hydrolysis in donkey liver.

Mitochondria, microsomes, and cytosol all hydrolyze carbamylphosphate. Microsomes show the greatest specific activity. The mitochondrial enzyme is localized in the inner membranes, has optimum pH 5.5 and is heat-stable; the cytosol enzyme has optimum pH 5.0 and is heat-labile. Km for carbamylphosphate is 1.1 to 1.2 X 10(-2 M for both the cytosol and the mitochondrial enzyme. Both enzymes are inhibited by high substrate concentrations.

Urea cycle enzymes in the liver of a dolphin Platanista indi.

Urea cycle enzymes are all shown to be active in dolphin liver. Acetylglutamate-independent cytoplasmic carbamylphosphate synthase is also present. Arginase is a basic protein, although less markedly basic than the dog enzyme. It is 118 per cent activated by heating at 50 degrees. Optimum pH is 10.5. Co++ and Ni++ inhibit the enzyme. AMP deaminase, glutamicoxaloacetic transaminase, glutamate dehydrogenase and ornithine transaminase are also active in dolphin liver.

S-Adenosylmethionine decarboxylase in liver, heart and pancreas of pyridoxine-deficient chickens.

S-adenosylmethionine decarboxylase activity has been measured in liver, heart and pancreas of pyridoxine-deficient chickens: in liver and heart muscle it is increased, while in pancreas the activity is unchanged with respect to control animals. Insulin induced activity in liver and in heart muscle of normal as well as of pyridoxine-deficient chickens, while in the pancreas an induction was observed in the control animals and a decrease in the deficient ones. These data appear to rule out any involvement of pyridoxal phosphate in the reaction catalyzed by S-adenosylmethionine decarboxylase.

Catabolism of ornithine in chicken liver.

It is shown that most ornithine in a chicken liver homogenate is decarboxylated in the particulate fraction. This fraction, however, requires the cytosol for complete activity. The dialyzed supernatant does not activate decarboxylation of ornithine, while the supernatant is more effective when previously inactivated at 100 degrees C. The supernatant can be substituted by the intermediates of the citric acid cycle (oxaloacetate, citrate, succinate, malate), by pyruvate, and partially by ADP as well. Rotenone blocks decarboxylation suggesting that this occurs through the pathway ornithine leads to glutamic semialdehyde leads to glutamate leads to alpha-ketoglutarate, which in turn is decarboxylated. The activating metabolites would thus have a role in reoxidizing NADH, and the ketoacids also in supplying the acceptor for transamination of glutamate, and indirectly for ornithine transamination. Pyruvate and oxaloacetate do not transaminate with ornithine. Insulin promotes a marked increase of cytosol ornithine decarboxylase activity, but has little effect on decarboxylation by the particulate cellular fraction.

Effect of caffeine on ornithine metabolism in rat brain, liver and kidney.

Prolonged treatment with caffeine promotes in rats an increase of liver ornithine carbamyltransferase activity (14-day treatment). In contrast, arginase activity is already reduced in brain and kidney after 10 days, and in the liver much later (17 days). Ornithine transaminase activity was increased in both liver and kidney, while in the brain it was reduced (17 days). Ornithine decarboxylase activity showed only minor modifications in kidney, while it was unchanged in brain. Of the polyamines, only spermidine was significantly modified, being increased in brain, decreased in liver and kidney. Although these results do not explain the mechanism of the modification of brain arginine and ornithine concentration promoted by caffeine, they point to further marked effects, i.e. on OAT activity and on spermidine concentration, which could have a relevant metabolic role.

Distribution and function of polycystin-2 in mouse retinal ganglion cells.

The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.