Salvatore Macaione

Serum Ionized Magnesium Levels in Type 2 Diabetic Patients with Microalbuminuria or Clinical Proteinuria

Background/Aims: The association between microalbuminuria and magnesium depletion is a controversial issue, and serum ionized magnesium levels have not been previously studied in patients with different grades of diabetic nephropathy. Therefore, the aim of this study was to evaluate circulating ionized magnesium concentrations in patients with non-insulin-dependent diabetes mellitus (NIDDM) and incipient or overt diabetic nephropathy. Methods: We measured fasting plasma glucose, creatinine, creatinine clearance estimate, total cholesterol and triglycerides, and serum ionized magnesium (ion-selective electrodes, ISE) in 30 NIDDM patients with urinary albumin excretion rate (UAER) <20 μg/min (normoalbuminuria), 30 NIDDM patients with microalbuminuria (20 < UAER < 200 μg/min), 30 NIDDM patients with clinical proteinuria (UAER >200 μg/min), and 20 healthy subjects. Results: Serum ionized magnesium levels were significantly reduced in diabetic patients when compared to control subjects (0.39 ± 0.06 vs. 0.58 ± 0.05 mmol/l, p < 0.001). Moreover, diabetic patients with microalbuminuria or clinical proteinuria showed a significant decrease in serum ionized magnesium with respect to normoalbuminuria group (normoalbuminuria: 0.45 ± 0.02 mmol/l; microalbuminuria: 0.36 ± 0.05 mmol/l, p < 0.001; clinical proteinuria: 0.35 ± 0.04 mmol/l, p < 0.001). Serum ionized magnesium showed a significant negative correlation with plasma HbA1c and triglycerides in both microalbuminuria and clinical proteinuria groups. Multiple linear regression analysis showed that circulating ionized magnesium levels decrease together with the increase of plasma HbA1c and triglycerides in NIDDM patients with incipient or overt nephropathy, also after adjusting for age, sex, BMI, diabetes duration, systolic and diastolic blood pressure, hypoglycemic therapy, plasma creatinine, creatinine clearance, plasma cholesterol and fasting glucose. Conclusions: Microalbuminuria and clinical proteinuria, as well as poor glycometabolic control and hypertriglyceridemia, are associated to relevant alterations in magnesium metabolism, and the measurement of serum ionized magnesium seems to represent a useful biochemical tool for the study of magnesium disturbances in patients with different grades of diabetic nephropathy.

Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina.

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non‐NMDA‐agonists, such as kainic acid (KA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7–8% of total retinal LDH was released after exposure to a 50 µm concentration of non‐NMDA agonists. These values were similar to those obtained after 100 µm NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 µm) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase‐3‐like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time‐related appearance of TUNEL‐positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 µm concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.

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.

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 Lockes 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.

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.

Localization of GABA system in rat retina

GABA is a known constituent of the retina and choroid (but not of iris or ciliary body) in the dog and ox (KOIIMA, MIZUNO and MIYAZAKI, 1958); it has been isolated from the eyes of mouse and chicken embryos as well as from adult frog retinae (KURIYAMA, SISKEN, HABER and ROBERTS, 1968). In the retina GABA can originate from glucose (CATANZARO, CHAIN, POCCHTARI and READING. 1962) and glutamate (CHAIN, POCCHIARI and READING, 1962); this metabolite, however, has not been detected in the optic nerve of the dog (ROBERTS, LOWE, GUTH andJELINEK, 1958), ox (FLOREY, 1960) and rabbit (KURIYAMA et al., 1968), even though the latter proved to have a slight glutamate decarboxylase (GAD) activity. GABA has an inhibitory influence on retinal electrical responses (AMES and POLLEN, 1969; KISHIDA and NAKA, 1967; KRAMER, SHERMAN and SEIFTER, 1967: SCHOLES and ROBERTS 1964) and the GABA content of the retina is diminished in the dark-adapted frog (GRAHAM, BAXTER and LOLLEY, 1970). Thus it seems probable that GABA has a significant function in the retina. The exact localization of the GABA system is not accurately known, but KURIYAMA et al. (1968) revealed high GABA and GAD levels in ganglion cells and lower levels in the receptor and bipolar cell layers of the microdissected rabbit retina. Our previous work on rat retina during postnatal growth (MACAIONE, CAMPISI and ALBANESE, 1970; MACAIONE, 1970) has shown that during the first month of life there is a double correlation between GABA content and GAD activity and also between GAD and y-aminobutyrate-a-oxoglutarate transaminase (GABA-T) activity. The variations in the GABA system between 10 and 21 days after birth are probably closely related to the maturation of neurons which differentiate during this period (MACAIONE, 1970; WEIDMAN and KUWABARA, 1969). To emphasize the significance and importance of retinal GABA we decided to carry out experiments upon localization of the GABA system in retinal cell layers, using the selective cytotoxic action of monosodium glutamate on the bipolar and ganglion cell layers and that of iodoacetate on the receptor cells of the rat retina.We have taken into consideration the probable presence of the GABA system in the pigmented epithelial layer, using ox eyes, because of the difficulty in obtaining a sufficient quantity of rat epithelial cellular elements.

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

Abstract: The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putres‐cine, spermidine, and spermine levels as well as ornithine decarboxylase, S‐adenosylmethionine decarboxylase, and N1‐acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adre‐nalectomized rats. At 6 h after corticosterone or dexameth‐asone administration, the specific activities of ornithine decarboxylase and N1‐acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S‐adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1‐acetylspermidine transferase activities in a dose‐dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses >5 mg/kg. The great increases in ornithine decarboxylase and N1‐acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1‐acetylspermidine transferase induction.

Cysteine oxidase activity in rat retina during development.

Abstract Cysteine oxidase activity has been determined in developing rat retina. Enzymic activity is present in 55, 000 × g supernate and in the crude mitochondria. Activity is rather low at birth; but increases with age; in mitochondria it reaches its maximum value at the 25th day while in supernate it increases more rapidly, reaching its maximum value 20 days after birth; unlike in the mitochondria, the activity of supernate considerably decreases during further development. The reason and significance of the postnatal changes in the mitochondrial cysteine oxidase activity are briefly discussed in relation with taurine formation.

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.

Amino acid levels in some brain areas of inducible nitric oxide synthase knock out mouse (iNOS −/− ) before and after pentylenetetrazole kindling

Inducible nitric oxide synthase knock-out (iNOS(-/-)) mice are valid models of investigation for the role of iNOS in patho-physiological conditions. There are no available data concerning neuroactive amino acid levels of iNOS(-/-) mice and their behaviour in response to pentylenetetrazole (PTZ). We found no significant differences in the convulsive dose 50 (CD(50)) between iNOS(-/-) and control (iNOS(+/+)) mice, however, iNOS(-/-) mice reach the kindled status more slowly than control, suggesting that in basal condition the GABA-benzodiazepine inhibitory inputs are unaltered by iNOS mutation. Clear differences between iNOS(+/+) and iNOS(-/-) mice amino acid concentrations were evident both in basal conditions and after kindling. Our results show that aspartate was significantly lower in all brain areas studied except the brain stem whereas glutamate and glutamine were significantly higher in the cortex, hippocampus and brain stem. GABA was slightly and not significantly higher in the cortex, hippocampus and brain stem, whereas taurine was significantly higher in all areas except diencephalon and glycine was significantly lower in the diencephalon and cerebellum. In this context, the inability of iNOS(-/-) mice to increase the NO levels following PTZ administrations indicate that NO might play a pro-epileptogenic role in the genesis and development of some types of epilepsy. Since there is no correlation between neurotransmitter levels and the development of kindling, it is possible to exclude that the difference between the two strains is due to an imbalance between the considered neurotransmitters, and it is then possible that this difference is due to the presence of iNOS, which might be involved in long term plasticity of the brain.