Nitric oxide synthase in chick embryo retina during development.

High levels of nitric oxide synthase were found in the early stages of developing chick embryo retina. The enzyme activity sharply decreased up to 13-day-old chick embryo retina, when the level of the last embryonic day was reached. The results show that nitric oxide is synthesized in chick embryo retina prior to synaptogenesis. The incubation of chick embryo retinas in presence of NMDA increased the synthesis of nitric oxide, thus, the appearance of nitric oxide production before the synaptogenesis in the retina as well as in the brain may be considered as signal for the development and shaping of neuronal and non-neuronal cells.

Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells.

The stimulation of NMDA receptor increased [3H]GABA release from preloaded cultured retina cells. This effect appears to be mediated by NO production, since addition of L-NA reduces NMDA-evoked [3H]GABA release. Spermine/NO complex, an NO donor, mimics the effect produced by NMDA. The addition of zaprinast, a phosphodiesterase inhibitor, as well as 8-Br-cGMP enhances the NMDA-evoked [3H]GABA release. These results agree with the existence in chick retina cells of NO/cGMP pathways and support a role for NO in NMDA-evoked events. The activation of this receptor complex through maturative stages of the retina together with the NO-mediated increase in GABA release may account for NMDA differentiative effect in culturing retina cells.

NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells.

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells. These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure. These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.

Changes in plasma, erythrocyte, and platelet magnesium levels in normotensive and hypertensive obese subjects during oral glucose tolerance test.

We evaluated the 75-g oral glucose tolerance test (OGTT)-induced modifications in glucose, insulin, and norepinephrine plasma concentrations, and in plasma, erythrocyte, and platelet magnesium levels in two groups of obese subjects (normotensive obese, NT-Ob, N = 19; hypertensive obese, HT-Ob, N = 15), and in a group of healthy control subjects (N = 12). During OGTT we detected a reduction in plasma magnesium concentrations and an increase in erythrocyte and platelet magnesium levels in the controls, whereas in both normotensive and hypertensive obese subjects, there was a reduction in plasma, erythrocyte, and platelet magnesium levels. Furthermore, no statistically significant difference was detected among the groups studied as regards delta-plasma magnesium. On the other hand, delta-erythrocyte magnesium and delta-platelet magnesium were negative in the NT-Ob (delta-erythrocyte magnesium: -0.24+/-0.08 mmol/L; delta-platelet magnesium: -0.49+/-0.09 micromol/10(8) cells) and HT-Ob (delta-erythrocyte magnesium: -0.20+/-0.10 mmol/L; delta-platelet magnesium: -0.50+/-0.11 micromol/10(8) cells) groups, and positive in control subjects (delta-erythrocyte magnesium: 0.40+/-0.08 micromol/L; delta-platelet magnesium: 0.47+/-0.09 mmol/ 10(8) cells). Finally, a direct correlation was found between delta-norepinephrine and delta-erythrocyte magnesium (r = 0.80, P < .01) in the control group, and a negative correlation was detected between delta-norepinephrine and delta-platelet magnesium (r = -0.58, P < .05) in the HT-Ob group. Our results seem to indicate that the insulin resistance status, the hyperglycemia, and the disregulation of the adrenergic system in obese subjects could be involved in the pathogenesis of the magnesium homeostasis impairment observed in the obese subjects.

The localization of phenolic and tyrosyl ring thyroxine deiodination in rat and mouse retina.

To elucidate T(4) metabolism in various cell types of rat retina, 5-monodeiodinating and 5?-monodeiodinating activities were studied in retinal cell layers obtained by selective cytotoxic action of monosodium glutamate on bipolar and ganglion cell layers and by iodoacetate effect on photoreceptor cells. Concomitantly these enzyme activities were studied in C3H/HeN mouse retina genetically deprived of photoreceptor cells. Deiodinase activities were low in rat and mouse retina deprived of photoreceptors. The 5?-monodeiodination rate of T(4) was higher than T(4) tyrosyl ring deiodination in cell layers examined and the highest values were found in the photoreceptor cells. Data support the hypothesis that phenolic and tyrosyl ring deiodinase activities are present in the photoreceptor cells. Their reciprocal changes may regulate the nuclear function which in turn controls the rhythmical renewal of rod outer segments.

Glutamate-induced increases in transglutaminase activity in primary cultures of astroglial cells.

Glutamate exposure of astroglial cells caused ligand-gated channel receptor activation, associated with excitotoxic cell response. We investigated the effects of 24 h glutamate exposure on transglutaminase in astrocytes primary cultures at 7, 14, and 21 days in vitro (DIV). Increases in enzyme activity were observed as a function of cell differentiation stage in glutamate-treated cultures. These effects were significantly reduced when GYKI 52466, an AMPA/KA receptors inhibitor, was added to the culture medium prior to incubation with glutamate. Microscopy observation on transglutaminase-mediated, fluorescent dansylcadaverine incorporation in living cells was consistent with these results. Western blotting analysis with monoclonal antibody showed that glutamate also up-regulated tissue transglutaminase expression, which reached the highest values in 14 DIV cultures. Confocal laser scanning microscopy analysis of immunostained astroglial cells showed a mainly cytoplasmic localisation of the enzyme both in control and treated cultures; nevertheless, counterstaining with the nuclear dye acridine orange demonstrated the presence of tissue transglutaminase also into the nucleus of glutamate-exposed and 21 DIV cells. The increases in enzyme expression and localisation in the nucleus of glutamate-treated astroglial cells may be part of biochemical alterations induced by excitotoxic stimulus.

Nitric oxide enhances amino acid release from immature chick embryo retina.

Nitric oxide (NO) was investigated for its ability to induce amino acid release from immature chick retina. The production of endogenous NO by activation of NO synthase after stimulation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptor caused a significant increase in basal release of gamma-aminobutyric acid (GABA) and glutamine, whereas a more modest increase in the glutamate release was also observed. The exposure of chick retina from 9-day-old embryos to NO-generating compounds, S-nitroso-N-acetylpe-nicillamine (SNAP) and sodium nitroprusside (SNP) produced a dose dependent increase in GABA, glutamine, and glutamate release. This effect was reduced by about 80% by haemoglobin. These results indicate that NO has a stimulatory effect on amino acid release from chick embryo immature retina. However, this effect does not appear to involve a cGMP-related mechanism because 8-bromo-cGMP, a stable analogue of cGMP, failed to affect spontaneous amino acid release and because zaprinast did not enhance NMDA-stimulated release. In conclusion, our present observations may account for a role of NMDA-mediated events in the biochemical maturation under depolarizing conditions.

beta-Endorphin enhances polyamine transport in human lymphocytes.

Opioid peptides, such as beta-endorphin (beta-end), are capable of modulating in vitro proliferative response of lymphocytes. We attempted to determine the role of extracellular polyamines in the regulation of immune responses to opioid peptides by measuring the extent of polyamine uptake as adaptional response to cell activation. beta-end dose-dependently enhanced the incorporation of radioactive spermidine and spermine. When the cells were depleted of spermidine, with addition of specific inhibitors of both biosynthesis and interconversion pathway, a large increase in the incorporation of radioactive spermidine was observed. This effect appeares to be specific for beta-end, although a non-opiate-specific receptor could be involved, since beta-end-enhanced incorporation of radioactive spermidine is not blocked by naloxone. We conclude that the enhancement of polyamine incorporation may be considered as an integral component of lymphocyte activation by beta-end.

Taurine distribution in rat tissues during development.

1. Taurine levels have been determined in eight rat organs. 2. During postnatal growth the taurine content in retina, heart, small intestine, spleen and lung increases with advancing age, although adult values are not reached at the same time. 3. In contrast the taurine content decreases with age in brain cortex, liver and kidney. 4. The taurine in subcellular fractions of adult, 20-day-old and 5-day-old rat tissues exists predominantly in the cytosol of the cell. Taurine content in particulate fractions shows marked variations during development in the different organs. 5. Taurine distribution in the subcellular fractions suggests that some of the cellular taurine in the tissues is not freely mobile in cytosol.

Thyroxine effects on polyamine metabolism in rat cerebellum and brain cortex during postnatal development.

Polyamine content, ODC and SAMDC activities have been assayed in cerebellum and brain cortex of hyperthyroid rats during postnatal development. Daily thyroxine treatment induced ODC and SAMDC biosynthesis in early periods of postnatal life in both cerebellum and brain cortex. In addition, in comparison to controls an increase in spermidine and spermine content was shown to occur in hyperthyroid rats. A functional correlation between polyamine content and nucleic acids could explain a correlation between polyamine biosynthesis and morphofunctional maturative processes in the brain.

Methionine activating enzyme in various human platelet populations.

Human platelets have been separated into four populations by a discontinuous sucrose gradient. MAE activity has been determined in the various platelet populations and significant differences were obtained with respect to platelet size, the large-heavy platelets showing a higher enzymatic activity than the small-light ones. These data suggest that higher MAE activity in the younger and large-heavy platelets may be responsible for a much higher biochemical and functional efficiency.

Subcellular distribution of pyridoxal kinase in ox retina.

PL kinase activity has been determined in primary and secondary subcellular fractions of ox retina. Enzymic activity is predominantly located in the soluble fraction (S3). About 65% of the recovered PL kinase activity of crude mitochondria is released from synaptosomal fraction after hypoosmotic treatment. PL kinase activity in supernatant fraction (S3) does not exceed the enzyme activity measured in the homogenate, excluding the presence of a particulate-bound inhibitor to PL kinase in the homogenate. The possible physiological significance of PL kinase cellular compartmentation has been considered.

Effects of estrogens and progesterone on GABA system in ovariectomized rat retina.

In an attempt to characterize the effect of estrogens and progesterone on retinal GABA metabolism, female Wistar Nossan rats were ovariectomized and treated for three days with 17 beta-estradiol (1 microgram/day), estrone (2 micrograms/day), estriol (200 micrograms/day) and progesterone (500 micrograms/day) or vehicle. After 3 days of steroid hormones treatment, GAD, GABA-T activities and GABA content were measured in retina homogenate. Progesterone did not reduce GAD, GABA-T activities and GABA content from ovariectomized levels. 17 beta-estradiol, estrone and estriol decreased the GAD activity. Furthermore the decrease in GAD activity was maximal for 17 beta-estradiol whereas the estrogens treatment was ineffective on GABA-T. GABA content was significantly decreased only by 17 beta-estradiol. Estrogens reduced the Vmax of GAD for glutamate as a substrate without changing the Km.

Cysteic and cysteine sulphinic acid decarboxylase activity in rat tissues during postnatal growth.

1) Cysteic and cysteine sulphinate decarboxylase activities have been determined in rat retina, brain, liver, kidney, lung and spleen during postnatal growth. 2) The two enzymic activities increase as function of age in retina, brain and liver. 3) In contrast the cysteic and cysteine sulphinate decarboxylase activities decrease with age in kidney, lung and spleen. 4) The rate of decarboxylation of cysteine sulphinate is higher than cysteic acid and the ratio of the two decarboxylating activities for each tissue is constant; this uniformity may exclude the existence of two separate enzymes.

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.

Putrescine, polyamines, and N1-acetylpolyamine levels in retina, visual cortex and cerebellum of free-running mice kept under continuous light or darkness.

Putrescine, spermidine and spermine levels were detected in the retina, visual cortex, cerebellum and parietal cortex of CD1 mice exposed to 36h continuous light or darkness. Retinal putrescine and polyamine concentrations were found to be highest in dark-adapted mice, and the stimulation of dark-adapted retina with flicker illumination was also accompanied by a significant decrease in putrescine, spermidine and spermine levels. In visual cortex as well as in cerebellum spermidine and spermine contents were higher in dark-adapted mice in comparison to light-exposed animals, while in parietal cortex no significant change was found neither in spermidine nor spermine levels. In the brain areas studied flicker illumination produced no significant decreases in putrescine and polyamine contents. The total polyamines expressed as putrescine equivalents were noticeably decreased in retina, visual cortex and cerebellum of light-adapted mice. In the retina spermine/spermidine molar ratio was significantly higher than in dark-adapted mice. The administration of N1, N2-bis-(2,3-butadienyl)-1,4-butanediamine (MDL 72527) produced a strong decrease of retinal putrescine and spermidine concentrations in both dark-adapted and light-exposed mice, and in the retina of mice exposed to continuous light a significant decrease in the spermine level was also observed. According to the influence on polyamine reutilization, after the irreversible inhibition of polyamine oxidase by MDL 72527, in the retina N1-acetylspermidine and N1-acetylspermine accumulation was highest in light-adapted mice. On the contrary in visual cortex, cerebellum and parietal cortex the MDL 72527 administration produced a more marked decrease of putrescine and spermidine contents in mice kept in continuous darkness.

A decrease in guanylate cyclase activity in some tissues of C3H/HeJ mice with retinal dystrophy.

Guanylate cyclase activity was assayed in homogenates, in particulate and soluble fractions from retina, cerebellum, cerebral cortex and adrenal gland of adult C3H/HeJ mice with a dystrophic retinopathy. In comparison to control mice (DBA/1J), in C3H/HeJ strain a significant decrease in guanylate cyclase activity occurred in homogenates from retina, cerebellum and adrenal gland. In particular a significant decrease was found in particulate fraction of retina, in the soluble fraction of cerebral cortex and cerebellum and in both fractions of the adrenal gland. In contrast to the retina and cerebellum where guanylate cyclase activity in homogenates was found significantly decreased both in the male and female, in the cerebral cortex guanylate cyclase decreased in both sexes although in female this was more marked.

Platelet magnesium depletion in normotensive and hypertensive obese subjects: the role of salt-regulating hormones and catecholamines.

We measured plasma and platelet magnesium concentrations, plasma epinephrine and norepinephrine, and plasma aldosterone and renin concentrations in normotensive (NT-Ob, n = 19, BMI 35.7 +/- 7.4 kg/m2, WHR 0.92 +/- 0.05) and hypertensive (HT-Ob, n = 11, BMI 35.2 +/- 3.6 kg/m2, WHR 0.93 +/- 0.07) obese subjects, and in a group of age- and sex-matched lean controls (n = 14, BMI 23.1 +/- 1.8 kg/m2, WHR 0.79 +/- 0.05). Plasma aldosterone and renin concentrations were significantly higher in obese subjects with respect to controls. Moreover, plasma norepinephrine and epinephrine levels were significantly increased in obese subjects, and plasma norepinephrine was higher in HT-Ob when compared to NT-Ob group. Platelet magnesium concentrations were significantly reduced in both normotensive and hypertensive obese subjects with respect to controls (controls 2.65 +/- 0.35 mumol/10(8) cells, NT-Ob 2.02 +/- 0.19 mumol/10(8) cells--p < 0.001, HT-Ob 1.98 +/- 0.18 mumol/10(8) cells--p < 0.001), while a slightly significant decrease in plasma magnesium levels was only detectable in HT-Ob group. Urinary magnesium and magnesium fractional excretion were significantly increased in hypertensive obeses. Pearson's correlation analysis, separately performed in each group of subjects, showed that plasma aldosterone, renin, epinephrine, norepinephrine and magnesium fractional excretion were negatively correlated to platelet magnesium levels in NT-Ob and HT-Ob groups, but not in lean controls. The multiple linear regression analysis performed in the whole group of obese subjects considering platelet magnesium as a dependent variable showed that platelet magnesium decrease together with the increase in plasma epinephrine (p = 0.046) and norepinephrine (p = 0.020), also after adjusting for age, sex, BMI, WHR, HOMA IR and diagnosis of hypertension. Furthermore, platelet magnesium showed a trend for negative association (p < 0.1) to plasma aldosterone and magnesium fractional excretion in multivariate analysis. The impairment in platelet magnesium handling observed in normotensive and hypertensive obese patients seems to be associated to a rise in renin-angiotensin-aldosterone and sympathetic systems activity. Our results suggest that platelet magnesium depletion, together with disturbances of salt-regulating hormones and catecholamines, may be involved in the pathophysiology of cardiovascular complications from obesity.

Plasma (total and ionized), erythrocyte and platelet magnesium levels in renal transplant recipients during cyclosporine and/or azathioprine treatment.

We evaluated total and ionized plasma magnesium levels, and erythrocyte and platelet magnesium concentrations from two groups of renal transplant recipients treated either with cyclosporine, azathioprine and prednisolone (group CAP, n = 8) or with azathioprine and prednisolone (group AP, n = 13), and in a group of age- and sex-matched healthy subjects (n = 10). Reduced plasma (total and ionized), erythrocyte and platelet magnesium concentrations were found in both CAP and AP groups with respect to controls (CAP: total plasma Mg median 0.61 vs 0.86 mmol/L, p < 0.01, ionized plasma Mg median 0.43 vs 0.58 mmol/L, p < 0.001, erythrocyte Mg median 2.18 vs 2.56 mmol/L, p < 0.05, platelet Mg median 1.75 vs 2.84 mmol/10(8) cells, p < 0.001; AP: total plasma Mg median 0.62 vs 0.86 mmol/L, p < 0.01, ionized plasma Mg median 0.48 vs 0.58 mmol/L, p < 0.001, erythrocyte Mg median 2.30 vs 2.56 mmol/L, p < 0.05, platelet Mg median 1.75 vs 2.84 mumol/10(8) cells, p < 0.001), while no difference was found between the two groups of transplant recipients as regards plasma and intracellular magnesium levels. Magnesium fractional excretion was higher in transplant recipients than in the control group (Mg fractional excretion median AP 18.6 per cent and CAP 12.8 per cent vs controls 3.5 per cent), whereas no difference was found between patients and control subjects for urinary magnesium 24h excretion. Moreover, in the whole group of transplant recipients (n = 21), urinary magnesium showed an inverse correlation with platelet (rs = -0.54, p < 0.05) and ionized plasma magnesium (rs = -0.48, p < 0.05), and time after transplantation showed a negative correlation with platelet magnesium concentrations (rs = -0.73, p < 0.001), and a direct correlation with fractional magnesium excretion (rs = 0.53, p < 0.05). Finally, a direct relationship between platelet magnesium and ionized plasma magnesium was also detected in the whole group of transplant recipients (rs = 0.47, p < 0.05). Both intraplatelet magnesium depletion and ionized plasma magnesium reduction induced by immunosuppressive therapy could be involved in the increased risk from atherosclerotic disease in renal transplant recipients.

[Obesity and hypertension: the role of magnesium]. / Obesità ed ipertensione: ruolo del magnesio.

We focus on the recent "ionic hypothesis", in which an alteration of ionic metabolism represents a peculiar event in the pathogenesis of obesity and hypertension. We report the results from our original studies in which we evaluated intraplatelet magnesium levels. In a study on normotensive and hypertensive patients with non insulin-dependent diabetes mellitus and healthy control subjects, we showed a common reduction of plasma, erythrocyte and platelet magnesium levels in both normotensive and hypertensive diabetics with respect to control. Anyway, hypertensive diabetics showed a greater reduction of intraplatelet magnesium concentrations when compared to normotensive diabetics. Using the same technique, we found reduced erythrocyte and platelet magnesium concentrations in patients with essential hypertension with respect to the control group. In a successive study, we found, in the group of normotensive obese, that erythrocyte and platelet magnesium levels were significantly lower than those of the control group, while in hypertensive obese patients a reduction of plasma magnesium levels has been also detected. In conclusion, in these studies has been confirmed the existence of a reduction of the intracellular magnesium concentrations, which is common in hypertensive and obese patients.