Sulfatides and arylsulfatase A activity in major salivary glands of hamster (Mesocricetus auratus) after adenocarcinoma induction in oral cavity.

A biochemical study of sulfatides and arylsulfatase A (ASA) was carried out in the submandibular and sublingual glands of the male and female hamster Mesocricetus auratus after experimental induction of oral adenocarcinoma by 7,12-dimethylbenzanthracene (DMBA). Hamster experimental groups included control animals, animals treated with beta-carotene, animals treated with DMBA, and animals treated with DMBA plus beta-carotene. Oral cavity treatment with DMBA induced carcinogenesis in the buccal mucosa, but not in the major salivary glands, where nevertheless, the morphology and expression of both parameters examined changed. In fact, sulfatide concentrations and enzyme activity increased significantly, while in control and beta-carotene-treated hamsters they were similar in both glands and sexes. After administration of DMBA plus beta-carotene, sulfatide concentration decreased, as did ASA activity, slightly in the submandibular gland and remarkably so in the sublingual one of female hamsters. Thin-layer chromatography (TLC) analysis of lipid patterns, after DMBA treatment, revealed considerable differences, not only in sulfatides, but also in other lipid fractions, as well as between the two glands and two sexes. These findings show that oral cavity treatment with DMBA is not able to induce carcinogenesis in the major salivary glands examined; however, it does cause considerable metabolic changes.

Changes of retinal neurons and glial fibrillary acid protein immunoreactive astrocytes in spontaneously hypertensive rats.

OBJECTIVE: The influence of arterial hypertension on retinal neurons and glial fibrillary acid protein (GFAP) immunoreactive astrocytes was investigated in spontaneously hypertensive rats (SHRs). METHODS: The retinas of 4- and 6-month-old SHRs and age-matched Wistar-Kyoto rats (WKY) were investigated. A group of SHRs, treated from 4 to 6 months with the hypotensive drug hydralazine, was also examined. Microanatomical and immunohistochemical techniques associated with image analysis and the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) technique for apoptosis or necrosis were used, as well as astrocyte molecular biology (Western blot) techniques. RESULTS: In 4-month-old SHR and WKY rats, retinal morphology and the number of retinal neurons and of GFAP-immunoreactive astrocytes were similar, with the exception of the occurrence of 1% of TUNEL-positive ganglionic neurons in SHRs. In 6-month-old SHRs a decrease of retinal volume and of the number of ganglionic neurons and photoreceptors was observed, compared with age-matched normotensive WKY rats or younger SHR and WKY rats. Two per cent of ganglionic neurons and 5% of photoreceptors were also TUNEL positive. In 6-month-old SHRs, hypertrophic perivascular GFAP-immunoreactive astrocytes were found, whereas their number was unchanged compared to younger cohorts or WKY rats. An increased expression of GFAP was also noticeable in SHRs by Western blot analysis. Hypotensive treatment with hydralazine partly countered retinal changes occurring in SHRs. CONCLUSIONS: The occurrence of neuronal and astroglial changes when a stable hypertension was developed, and their sensitivity to antihypertensive treatment, suggest that they may represent a hypertension-related phenomenon.

Microanatomical changes of intracerebral arteries in spontaneously hypertensive rats: a model of cerebrovascular disease of the elderly.

Changes occurring in intracerebral arteries of 24-week-old spontaneously hypertensive rats (SHR) compared with age-matched normotensive Wistar-Kyoto (WKY) rats were assessed using microanatomical techniques associated with image analysis. Morphometric parameters investigated included arterial diameter, lumen area, wall area, and wall-to-lumen ratio. Intracerebral arteries (lumen diameter>46 microm) and arterioles (lumen diameter 46-10 microm) of frontal cortex, striatum, and hippocampus were examined. In frontal cortex of SHR arterial wall hypertrophy and luminal narrowing were observed. In striatum, an increase of wall area not accompanied by luminal narrowing predominates resulting in arterial hypertrophy without vasoconstriction. In hippocampal arteries of SHR, luminal narrowing, without changes of wall area was found indicating the occurrence of remodeling. In brain areas investigated, hypertensive changes affected primarily arterioles. The demonstration of a sensitivity of intracerebral arteries to hypertension suggests that changes of these vessels may represent a cause of brain structural alterations occurring in hypertension. The specificity of alterations occurring in intracerebral arteries of brain areas investigated may account for the different localization of cerebral lesions in cerebrovascular disease. The possibility that microanatomical changes developed in intracerebral arteries of SHR may represent a model of cerebrovascular disease of the elderly is discussed.

The hippocampus in spontaneously hypertensive rats: a quantitative microanatomical study.

The influence of hypertension on the morphology of hippocampus was assessed in spontaneously hypertensive rats of two, four and six months and in age-matched normotensive Wistar-Kyoto rats. Values of systolic pressure were slightly increased in two-month-old spontaneously hypertensive rats in comparison with age-matched Wistar-Kyoto rats and augmented progressively with age in spontaneously hypertensive rats. No microanatomical changes were observed in the hippocampus of spontaneously hypertensive rats of two months in comparison with age-matched Wistar-Kyoto rats, whereas a decrease of white matter volume was observed in the CA(1) subfield and in the dentate gyrus of four-month-old spontaneously hypertensive rats. In the hippocampus of six-month-old spontaneously hypertensive rats a reduction of grey matter volume both in the CA(1) subfield and in the dentate gyrus, a loss of neurons affecting to a greater extent the CA(1) subfield and an increase of glial fibrillary acid protein-immunoreactive astrocytes was found. The occurrence of apoptosis and/or necrosis identified using the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick end labelling technique was also observed in the CA(1) subfield and to a lesser extent in the dentate gyrus. The only change noticeable in the CA(3) subfield of six-month-old spontaneously hypertensive rats was a slight increase in the number of glial fibrillary acid protein-immunoreactive astrocytes. These findings indicate the occurrence of neuronal loss and of astrocyte changes in the hippocampus of spontaneously hypertensive rats of six months, being the CA(1) subfield the area most affected. The relevance of these neurodegenerative changes in hypertension and the possible occurrence of apoptosis and/or necrosis as expression of hypertensive brain damage is discussed.

Nephroprotective effect of treatment with calcium channel blockers in spontaneously hypertensive rats.

The influence of hypertension and of treatment with some dihydropyridine-type Ca(2+) channel blockers and with the nondihydropyridine-type vasodilator hydralazine on the morphology of kidney was investigated in 26-week-old spontaneously hypertensive rats (SHR) and in age-matched Wistar-Kyoto rats. Fourteen-week-old SHR were treated for 12 weeks with a nonhypotensive dose of lercanidipine or with equihypotensive doses of lercanidipine, manidipine, nicardipine, and hydralazine. In control SHR, systolic pressure values were significantly higher in comparison with Wistar-Kyoto rats. Treatment with the low dose of lercanidipine did not reduce systolic blood pressure in SHR, whereas the higher dose of lercanidipine or other compounds tested significantly decreased systolic pressure values. Glomerular hypertrophy accompanied by signs of glomerulosclerosis, increase of mesangial cells, and convoluted tubules degeneration were observed in control SHR. Hypotensive doses of Ca(2+) antagonists countered glomerular injury, the increase of mesangial cells, the reduction of capsular space, and tubular degeneration. Hydralazine, in spite of its hypotensive activity, displayed a slight nephroprotective action. The nonhypotensive dose of lercanidipine countered in part glomerular injury, narrowing of capsular space, and tubular degeneration, and decreased mesangial cell augmentation in SHR. These results suggest that treatment with dihydropyridine-type Ca(+2) antagonists counters hypertensive glomerular and tubular changes occurring in SHR. The demonstration of nephroprotection by the nonhypotensive dose of lercanidipine suggests that the renal effects of the compound may be in part unrelated to its hemodynamic activity.

Effect of calcium antagonists on glomerular arterioles in spontaneously hypertensive rats.

Through the use of microanatomic techniques, we investigated the effects of treatment with some dihydropyridine-type calcium antagonists (CAs) (ie, lercanidipine, manidipine, and nicardipine) and with the nondihydropyridine-type vasodilator hydralazine on hypertension-dependent glomerular injury and on the morphology of afferent and efferent arterioles in spontaneously hypertensive rats (SHR). Fourteen-week-old male SHR and age-matched normotensive Wistar-Kyoto rats were left untreated (control groups). Four additional groups of 14-week-old SHR were treated for 12 weeks with daily oral doses of 2.5 mg/kg lercanidipine, 5 mg/kg manidipine, 3 mg/kg nicardipine, or 10 mg/kg hydralazine. These treatments decreased systolic blood pressure values to a similar extent in SHR. Signs of glomerular injury, as characterized by glomerulosclerosis, hypertrophy, and an increased number of mesangial cells, were observed in control SHR. The treatment with CAs improved glomerular morphology and decreased the number of mesangial cells. Lercanidipine and manidipine were more effective than nicardipine in countering glomerular injury. In the SHR, both afferent and efferent arterioles revealed luminal narrowing, accompanied by increased wall thickness in efferent arterioles. The dihydropyridine-type derivatives that were tested decreased the luminal narrowing of afferent arterioles. Lercanidipine and manidipine countered the luminal narrowing of efferent arterioles. Hydralazine had no effect on hypertension-dependent glomerular injury or vascular changes. The present data indicate that lercanidipine and manidipine vasodilate afferent and efferent arterioles in SHR. A vasodilatory activity on efferent arteriole, which is not induced by the majority of CAs, may represent an useful property in the treatment of hypertension complicated by renal disease.

Sulphatides in the brain of spontaneously hypertensive rats.

Sulphatides were assayed in preparations of frontal cortex, neostriatum and hippocampus of 6-month-old male spontaneously hypertensive rats (SHR, systolic pressure 215 +/- 6 mmHg) and age-matched normotensive Wistar-Kyoto (WKY) rats (systolic pressure 143 +/- 6 mmHg) by thin layer chromatography associated with spectrophotometry and histochemistry. The volume of gray and white matter of the above areas was also measured by microanatomical techniques associated with image analysis. Sulphatide levels were unchanged in the frontal cortex and neostriatum and decreased in the hippocampus of SHR in comparison with WKY rats. No changes of metachromatic sulphatide staining were found in the different brain areas investigated of SHR, whereas a decrease of positive metachromatic areas was noticeable in the frontal cortex and neostriatum, but not in the hippocampus of SHR. A reduction of volume of frontal cortex gray and white matter as well as of striosomes and of gray matter of hippocampus was found in SHR. No changes in the total volume of neostriatum and in the volume of white matter of hippocampus were observed between SHR and normotensive WKY rats. These findings, which are consistent with recent evidence of the occurrence of atrophic changes in the brain of SHR, showed that sulphatide levels were decreased in the hippocampus of SHR. In this area no reduction of white matter was observed. Sulphatide concentrations are thought to reflect the status of brain myelinated fibers. The not parallel decrease of sulphatide levels and white matter volume in the majority of brain areas investigated suggests the occurrence in SHR of sulphatide changes not corresponding simply to a reduction of myelinated pathways.

Forebrain white matter in spontaneously hypertensive rats: a quantitative image analysis study.

The volume and the morphology of brain white matter as well as the number and the size of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were investigated in 6-month-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats. The volume of frontal and occipital cortex and of hippocampus was decreased in SHR in comparison with normotensive rats, whereas the volume of neostriatum was unchanged. A remarkable decrease of the volume of internal capsule and striosomes, a moderate reduction of that of corpus callosum and no changes of the volume of external capsule and of white matter of hippocampus were also observed in SHR. In SHR the number of astrocytes was higher in the frontal and occipital cortex and in the white matter of the CA1 and CA3 subfields of the hippocampus, but not in the corpus callosum or in the grey matter of the CA1 and CA3 subfields. Staining for myelin did not reveal alterations in single fibre sheath morphology. These findings indicate the occurrence of changes of forebrain white matter in SHR, consisting in the reduction of it without qualitative modifications of myelinated fibres. The development of gliosis apparently not related with changes of volume of white matter was also found.

Autoradiographic localisation of muscarinic cholinergic receptor subtypes in human placenta.

The localisation of M1-M4 muscarinic cholinergic receptor subtypes was investigated in sections of normal human term placenta by light microscope autoradiography. Muscarinic cholinergic receptor subtypes were found almost exclusively in syncytiotrophoblast. Neither other cellular components of placenta, nor blood vessels were labelled. Quantitative analysis of the density of silver grains developed in sections incubated with the different protocols for labelling M1-M4 receptor subtypes, revealed that syncytiotrophoblast expresses all subtypes of muscarinic cholinergic receptor investigated. A higher density of binding sites was found in the apical than in the basal portion of syncytiotrophoblast. The demonstration of muscarinic cholinergic receptors in syncytiotrophoblast suggests that a cholinergic system may have a role in regulating transport of compounds from maternal to foetal interface.

Arylsulphatase A activity and sulphatide concentration in the female rabbit oviduct are under physiological hormonal influence.

Oviduct samples of female rabbits in different phases of the reproductive cycle were analysed to establish the role of sex steroid hormones in the regulation of sulphatide concentration and arylsulphatase A activity. In addition to biochemical procedures, histochemical techniques were used to localize both enzyme activity and the natural substrate. The plasma concentrations of progesterone and 17 beta-oestradiol were determined by radioimmunoassay (RIA). The findings show that the parameters examined undergo considerable changes during the different phases of the reproductive cycle. Oestrogens exert an inducing action on arylsulphatase A activity, while progesterone inhibits it. Fluctuations of the catabolic arylsulphatase activity condition the sulphatide concentration, which reaches maximum values at the eighth post-ovulatory day when progesterone dominance is consolidated. In this phase of the reproductive cycle, thin-layer chromatography confirms the presence not only of larger quantities of sulphatides, but also of all other lipid fractions.

Fluctuations in levels of various gangliosides in the different tracts of the oviduct of the frog (Rana esculenta) during the reproductive cycle.

The ganglioside content and pattern have been followed in the different tracts (rectus, convoluted and uterine) of the frog oviduct during the reproductive cycle. The main variations we observed are: a) average higher levels of ganglioside sialic acid in the preovulatory phase, with two peaks in March and April for the convoluted and rectus tract, respectively, and a more homogenous behaviour for the uterine tract; in all three tracts of the oviduct a minimum coincident with the ovulation has been found; b) a balanced presence of sulfolipids and gangliosides in the uterine tract: in fact sulfolipids, whose variations have been determined in a previous work, are higher when gangliosides are lower and vice versa, maintaining nearly constant the total negative charge due to these glycolipids; c) an alternate fluctuation of monosialo- and disialo-gangliosides in the preovulatory phase and a net trend toward the increase of monosialo- and the decrease of disialogangliosides in the postovulatory phase; trisialo-gangliosides are in general less represented and show less marked variations; d) the presence of particular gangliosides in particular moments of the reproductive cycle: Fuc-GM1, a fucosylated ganglioside, is higher than the more represented GM1 during the ovulation, while GD1 alpha, a ganglioside with a sialic acid residue linked to GalNAc, is steadily present in all three tracts after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid procedure for elimination of non-polar lipids hampering the usual polar lipid extraction and TLC separation.

A new and rapid method is proposed for extraction of non-polar lipids from tissues where they are present as abundant components which can interfere with the usual procedures of lipid extraction and TLC separation, and hamper, in particular, sulphatide visualization. A solvent more hydrophobic than chloroform, i.e. n-hexane, was utilized to remove the neutral lipids from samples of female rabbit parotid gland, and the n-hexane phase was used for TLC which showed considerable amounts of cholesterol esters, in addition to triglycerides, diglycerides and monoglycerides. The methanol phase, now devoid of non-polar lipids, was utilized to prepare TLC plates in order to separate and visualize the polar lipid fractions, in particular the sulphatides.

Expression of acidic glycosphingolipids and arylsulphatase A activity in human pathological endometrium.

Endometrium biopsic samples from women with cystic hyperplasia or adenocarcinoma were analysed by biochemical procedures to verify fluctuations in the acidic glycosphingolipid (sulphatide) concentration and arylsulphatase A (ASA) activity. Comparing the values of the considered parameters with those obtained in normal subjects, it was observed that ASA activity significantly increased in both pathologies; in contrast, sulphatide concentration underwent a non-significant decrease in hyperplasia and a statistically significant increase in neoplasia. The thin-layer chromatography (TLC) images revealed not only quantitative, but also qualitative differences in the lipid fractions. In fact, compared with controls, the sulphatides showed one more marked fraction in the neoplastic endometrium, and two fractions with different Rf values in the hyperplastic one. Moreover, two new unknown fractions also appeared in some subjects with cystic hyperplasia. The findings suggest the lipid metabolism undergoes considerable changes under the pathological conditions examined. The fluctuations observed, in particular, in the sulphatide concentration are believed to be related to changes in the biosynthetic and catabolic activities of the key enzymes directly involved in their metabolism, i.e. arylsulphatase A and sulphotransferase, which are regulated by sex hormones.

Comparative analysis of lysosomal hydrolases and natural glycoprotein substrates in the rat major salivary glands.

1. The activities of some lysosomal hydrolases and the concentrations of their natural substrates were studied in the submandibular and sublingual glands of male and female rats using biochemical procedures. 2. In sublingual gland enzyme activities and substrate concentrations show the highest values. 3. The enzyme activities appear, in general, lower and the natural substrate concentrations higher in the females with respect to males. 4. In both glands beta-galactosidase shows the highest activity and beta-glucosidase the lowest. 5. These findings suggest that metabolic turnover of glycoproteins is slower in females than in males, probably because the oestrogens control the activity of lysosomal hydrolases.

Changes induced by progesterone treatment in the sulphatides of the frog oviduct.

Sulphatides have been studied by histochemical and biochemical procedures in the oviduct of the frog in different experimental conditions. In ovariectomized or hypophysectomized animals, compared to sham-operated, an increase in sulphatides was observed. The progesterone treatment did not significantly modify this lipid fraction in ovariectomized frogs, while in hypophysectomized frogs it induced a further increase. Densitographic profiles of the sulphatides, obtained by thin-layer chromatography (TLC) and also recorded by Tesak equipment, were similar in ovariectomized or hypophysectomized frogs following hormone treatment because they showed three distinct fractions in both experimental groups of animals. The appearance of a third fraction never previously observed was probably induced by the progesterone treatment. Moreover, under the effects of this hormone, the phospholipid fractions (phosphatidylcholine and phosphatidylethanolamine) also showed different densitographic profiles.

Sialic acid and neuraminidase activity in the frog oviduct: comparative biochemical investigation in the different tracts during the reproductive cycle.

1. A biochemical study was carried out on the protein-bound and lipid-bound sialic acid, and neuraminidase activity in the different tracts of the oviduct of the frog Rana esculenta during the reproductive cycle. 2. Plasma sexual steroids were also investigated by RIA. 3. Fluctuations in neuraminidase activity are related to that of glycoprotein sialic acid and plasma estradiol. Glycolipid sialic acid does not have a close relationship either with neuraminidase or plasma estradiol. 4. Very high plasma concentration of progesterone before ovulation and, on the contrary, its drop after ovulation were observed. 5. The results are discussed and hypotheses advanced to explain fluctuations of the studied parameters during the reproductive cycle.

Arylsulphatase activity in the oviduct of the frog Rana esculenta. II. Progesterone-induced changes following ovariectomy and hypophysectomy.

The effects of progesterone treatment on arylsulphatase activity were studied histochemically and biochemically in the frog oviduct under different experimental conditions. In ovariectomized animals, the hormone induced a large increase in enzyme activity, while in hypophysectomized ones there was a large decrease in this activity. These results indicate that the facilitatory and inhibitory effects of progesterone on arylsulphatase activity are influenced by the presence in situ of the gonad. Hypotheses are advanced to explain different effects of the progesterone treatment.

Arylsulphatase activity in the oviduct of the frog Rana esculenta. I. Oestradiol-induced changes following ovariectomy and hypophysectomy.

The effects of oestradiol treatment on arylsulphatase activity in the frog oviduct are reported. Oestradiol-induced changes were also investigated in ovariectomized and hypophysectomized animals. Under all the experimental conditions, hormonal treatment causes an increase in enzyme activity. This can be observed biochemically and also histochemically on frozen sections. Hypotheses are advanced to explain fluctuations in arylsulphatase activity.

Arylsulphatase activity and cerebroside sulphates in the frog oviduct during the reproductive cycle.

The presence of arylsulphatase A and cerebroside sulphates in different tracts of Rana esculenta oviduct during different phases of the reproductive cycle were investigated by histochemical and biochemical procedures. The results indicate that enzyme activity shows seasonal fluctuations connected with the phase of the sexual cycle. The concentrations of cerebroside sulphates (the natural substrates of arylsulphatase A) is related to the activity of this hydrolytic enzyme. The role of arylsulphatase A activity in regulating the substrate concentration, and particularly that of sulphatides, is discussed.