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Epidermal growth factor (EGF) and other EGF-related growth factors, such as transforming growth factor-α, are able to stimulate neuroblastoma (NB) cell proliferation. Epiregulin (Epi) is a growth factor belonging to the EGF family known to be more potent than EGF in mediating mitogenic signals. In this study, we tested the ability of Epi to stimulate a human NB cell line (SK-N-BE) proliferation. Surprisingly, Epi (50-1000 ng/ml) induced a reduction in SK-N-BE proliferation along with a morphological differentiation, associated with an increase in MMP-9 expression. Moreover, Epi-induced differentiation was inhibited by ERK1/2 phosphorilation inhibition. In conclusion, Epi could represent a novel and useful tool to oppose NB cell proliferation.

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

The morphology of cerebral cortex was investigated in male spontaneously hypertensive rats (SHR) aged 2, 4 and 6 months (pre-hypertensive, developing hypertension and established hypertension respectively) and in age-matched normotensive Wistar-Kyoto (WKY) rats using quantitative microanatomical techniques. Analysis included frontal and occipital cortex as a paradigm of motor and sensory cerebrocortical areas respectively. Values of systolic pressure were slightly higher in 2-month-old SHR compared to age-matched WKY rats and augmented progressively with increasing age in SHR. In frontal cortex of SHR a decrease of nerve cell number and of cortical volume was observed in layers V and VI of 4- and 6- month-old SHR, and in layers I-IV of 6- month-old SHR. In occipital cortex a decrease of the number of nerve cells and of cortical volume was observed in layers V and VI of 2-, 4-, 6- month-old SHR, and in layers I-IV of 6-month-old SHR. Numerical decrease of neurons in SHR affected to a greater extent occipital cortex than frontal cortex. An increase in the number of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes (hyperplasia) as well as in the mean immune reaction area (hypertrophy) was found in the two cerebrocortical areas investigated of 6-month-old SHR. The occurrence of apoptosis and/or necrosis identified using the terminal deoxyribo-nucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) technique was also observed in frontal and occipital cortex of 6-month-old SHR, but not of younger cohorts. These findings indicate the development of microanatomical changes in the cerebral cortex of SHR, the extent of which increases parallel with the progression of hypertension. The occurrence of cerebrocortical apoptosis and/or necrosis as well as the obvious astrogliosis occurring in established hypertension may account for the increased risk of vascular dementia that represents a specific trait of complicated hypertension.

Astrogliosis, consisting in astroglial proliferation and increased expression of the specific cytoskeletal protein glial fibrillary acid protein (GFAP) is common in several situations of brain damage. Arterial hypertension, which induces cerebrovascular changes, can cause also brain damage, neurodegeneration and dementia (vascular dementia). This study was designed to assess astroglial reaction in different brain areas (frontal cortex, occipital cortex, hippocampus and striatum) of spontaneously hypertensive rats (SHR) in the pre-hypertensive phase (2 months of age), in the developing phase of hypertension (4 months of age) and in established hypertension (6 months of age). SHR were compared to age-matched normotensive Wistar-Kyoto (WKY) rats. Analysis included reverse transcription-polymerase chain reaction (RT-PCR) of GFAP mRNA, GFAP immunochemistry (Western blot analysis) and immunohistochemistry. A significant increase of GFAP mRNA and an increase of GFAP immunoreactivity were noticeable in different brain areas of SHR compared to normotensive WKY rats at 6, but not at 2 or 4 months of age. Immunohistochemistry revealed a numerical augmentation (hyperplasia) and an increase in size (hypertrophy) of GFAP-immunoreactive astrocytes in frontal cortex, occipital cortex and striatum of SHR. In the hippocampus of SHR only a numerical increase of GFAP-immunoreactive astrocytes was found. These finding demonstrating the occurrence of astrogliosis in the brain of SHR with established hypertension suggest that hypertension induces a condition of brain suffering enough to increase biosynthesis and expression of GFAP similarly as reported in several neurodegenerative disorders and in brain ischemia.

Dihydropyridine (DHP)-type Ca2+ antagonists block primarily L-type Ca2+ channels and are used in the therapy of hypertension. They were also proposed for the treatment of several central nervous system disorders. In brain, these compounds bind both neuronal and vascular Ca2+ channels, but no studies have evaluated comparatively their density at neuronal and vascular level. This study has analyzed the pharmacological profile and the anatomical localization of L-type Ca2+ channels in rat frontal cortex, hippocampus and in forebrain pial and intracerebral arteries by radioligand binding assay and high resolution light microscope autoradiography. The DHP derivative [3H]nicardipine was used as a radioligand. Binding of [3H]nicardipine was consistent with the labeling of L-type Ca2+ channels. In frontal cortex, the highest density of binding sites was found in nerve cell body region, followed by the neuropil and the wall of intracerebral arteries. In hippocampus, the density of binding sites was higher in the nerve cell body region than in the neuropil of CA1, CA3, and CA4 subfields. In the dentate gyrus, a higher density of silver grains was developed in neuropil than in nerve cell body of granule neurons. With the exception of dentate gyrus, neuronal binding sites were more expressed than vascular binding sites in the hippocampus. In pial arteries [3H]nicardipine binding density decreased concomitant with the reduction of vessel diameter, whereas in intracerebral arteries [3H]nicardipine binding density displayed an opposite pattern. The above findings indicate that in brain the density of neuronal L-type Ca2+ channels was significantly higher than that of vascular ones. This may account for more pronounced neuronal than vascular effects after pharmacological manipulation of cerebral Ca2+ channels.

Retinal and choroids arteries changes were investigated ophthalmoscopically and with morphometric techniques in spontaneously hypertensive rats (SHR) of 26 weeks either untreated (control animals) or treated for 12 weeks equi-hypotensive doses of the Ca2+ antagonist nicardipine or of the non-dihydropyridine type vasodilatator hydralazine. Retinal and choroid arteries hypertensive changes were compared with those affecting pial and intracerebral arteries of frontal lobe. Ophthalmoscopic analysis revealed in control SHR a rarefaction of capillaries and a decrease of their length and area. Treatment with nicardipine and to a lesser extent with hydralazine countered ophthalmoscopic changes noticeable in SHR. Morphometric analysis revealed thickening of the wall and luminal narrowing of retinal, choroids, pial, and intracerebral arteries. Anti-hypertensive treatment decreased thickening of the arterial wall and increased luminal narrowing of different arteries investigated. Nicardipine was more effective than hydralazine in countering arterial hypertensive changes in SHR and displayed a vasodilatory activity on small sized retinal and cerebral arteries, that represent a vascular segment not sensitive to hydralazine. Comparative evaluation of the wall-to-lumen ratio revealed a similar pattern between retinal and intracerebral arteries, but not between other arteries investigated. This suggests that analysis of retinal arteries may be predictive of brain intracerebral arteries changes in hypertension.

The influence of hypertension and of treatment with the dihydropyridine-type Ca2+ antagonists lercanidipine, manidipine, nicardipine, and nimodipine and with non dihydropyridine-type vasodilator hydralazine on retinal nervous and glial fibrillary acidic protein (GFAP) immunoreactive astrocytes were investigated in male spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) were used as normotensive references group. Treatment of animals with oral equi-hypotensive doses of the above compounds started at 14 weeks of age and lasted for 12 weeks. Microanatomical analysis was extended to samples of frontal cortex and occipital cortex used as reference tissue. Different compounds investigated decreased to a similar extent systolic blood pressure values with the exception of nimodipine that in spite of the high dose used exerted a less pronounced hypotensive activity. Morphological changes including reduced thickness of retina and of inner plexiform, outer nuclear and layer of inner and outer segments plus outer limiting layer, and loss of ganglionic neurons were observed. GFAP-immunoreactive astrocyte hypertrophy was also found in control SHR. These phenomena were countered by treatment by treatment with dihydropyridine-type Ca2+ antagonists and to a lesser extent by hydralazine. The different Ca2+ antagonists tested exerted a similar protective effect on retinal, but not on brain neurons. The sensitivity of retina and cerebral cortex to anti-hypertensive treatment may be related to a different density of L-type Ca2+ channels in structures investigated or to kinetic reasons. The demonstration of a neuroprotective effect of Ca2+ antagonists on retina of SHR suggests that these compounds might protect to a some extent retina from hypertensive injury.

The effect of an eight-week treatment with the Ca2+ channel blocker nicardipine on different-sized pial arteries and intracerebral arteries was assessed in spontaneously hypertensive rats (SHR) by microanatomical techniques. Normotensive Wistar-Kyoto (WKY) rats were used as normotensive reference animals. In SHR a significant increase of systolic blood pressure (SBP) in comparison with WKY rats was noticeable. An increased thickness of tunica media and luminal narrowing were also seen in medium- and small-sized pial arteries, and in intracerebral arteries of SHR in comparison with WKY rats. The media-to-lumen ratio was also increased in medium (diameter between 150 and 50 microm) and small-sized (diameter < than 50 microm) pial and intracerebral arteries. Treatment with nicardipine significantly reduced SBP, the thickness of tunica media, media-to-lumen ratio and increased luminal area of medium- and small-sized pial arteries and of intracerebral arteries. These findings demonstrate that treatment of SHR with nicardipine induces a moderate vasodilatation of both pial and intracerebral arteries regulating cerebrovascular resistance. This property may be useful in avoiding generalized or exaggerated cerebrovascular dilatation in hypertension which could be accompanied by impaired brain perfusion.

Neurofilaments (NFP) are components of neuronal cytoskeleton involved primarily in axonal transport and in the regulation of dynamic activities of nerve cells. NFP consist of three subunits denominated high- (200 kDa, NFP-H), intermediate- (160 kDa, NFP-I), and low-molecular weight (68 kDa, NFP-L) neurofilament proteins. Their function and polymerization depends on phosphorylation status, and is regulated by Ca2+ influx. Ca2+ overload enhances degradation of NFP and may compromise axonal transport. An increased susceptibility to ischemia occurs in hypertension, which is also a cause of brain damage. In this study, the expression of phosphorylated NFP (P-NFP) was investigated in the brain of spontaneously hypertensive rats (SHR) using immunohistochemical techniques with antibodies against the phosphorylated epitope of NFP RT-97. Microanatomical analysis included frontal cortex, occipital cortex, hippocampus and cerebellar cortex. The effect of long-term treatment with the dihydropyridine-type Ca2+ antagonist nicardipine on the expression of P-NFP was investigated as well. In hypertension a decreased P-NFP immunoreactivity was observed in frontal and occipital cortex, in the CA1 subfield of hippocampus and in the dentate gyrus, but not in the CA3 subfield of hippocampus or in the cerebellar cortex. Treatment with a daily dose of 3 mg/kg of nicardipine and 10 mg/kg of hydralazine significantly reduced systolic pressure in SHR. The above dose of nicardipine and to a lesser extent a non-hypotensive dose of the compound (0.1 mg/kg/day), but not hydralazine, increased P-NFP immunoreactivity in the cerebral cortex and hippocampus, except the CA3 subfield. The possibility that rescued P-NFP immunoreactivity by treatment with nicardipine depends on improved brain perfusion caused by the compound and/or by countering neuronal Ca2+ overload is discussed.

The occupancy of L-type Ca2+ channels by treatment with an oral dose of the dihydropyridine-type Ca2+ antagonist nicardipine (sustained-release formulation) was evaluated in membrane preparations of rat frontal cortex and hippocampus using a radioligand binding assay technique, with [3H]-nicardipine as a ligand. Three hours after nicardipine administration, specific binding was decreased by about 15-20%, both in the frontal cortex and hippocampus. This indicates that oral nicardipine occupied approximately 15-20% of L-type Ca2+ channels. A progressive occupancy of Ca2+ channels was observed between six and 12 h after nicardipine administration. Twelve hours after drug administration, approximately 65-70% of Ca2+ channels were occupied. These findings indicate that oral treatment with 3 mg/kg of nicardipine (sustained-release formulation) occupies L-type Ca2+ channels in rat brain by more than 40% from the 6th to the 24th h after drug administration. This suggests that an oral dose of nicardipine (sustained-release formulation) in duces a significant occupancy of L-type Ca2+ channels in rat frontal cortex and hippocampus for about one day. The possible clinico-therapeutic relevance of this observation is discussed.

The influence of hypertension and of treatment with the dihydropyridine-type Ca+2 antagonist nicardipine on peripheral nerve vasculature were investigated in spontaneously hypertensive rats (SHR). Male SHR were treated from the 16th to the 26th week of age with vehicle (control group), with nicardipine, at the hypotensive dose of 3 mg/kg/day, or at the nonhypotensive dose of 0.1 mg/kg/day or with an equihypotensive dose (10 mg/kg/day) of the nondihydropyridine-type vasodilator hydralazine. Age-matched normotensive Wistar-Kyoto (WKY) rats were left untreated and used as normotensive reference animals. In SHR a significant increase of systolic pressure values accompanied by sciatic nerve microvascular changes, involving primarily interfascicular arteries and to a lesser extent intrafascicular arteries, was observed. Treatment with the hypotensive dose of nicardipine countered hypertension-dependent microvascular changes occurring in both interfascicular and intrafascicular arteries. The nonhypotensive dose of nicardipine and hydralazine displayed a modest activity on interfascicular arteries, but significantly countered hypertension-related changes involving intrafascicular arteries. The above findings indicate the occurrence of hypertension-related changes of peripheral nerve microvasculature and of positive effects induced by appropriate pharmacological treatment. Further work is in progress to identify the functional relevance of microanatomical observations of the present study.

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.

The influence of pregnancy-induced hypertension (pre-eclampsia) on muscarinic cholinergic receptors and on acetylcholinesterase (AChE) activity was investigated using frozen sections of the umbilical artery and vein. Pre-eclamptic patients undergoing Caesarean delivery and normotensive pregnant control woman undergoing Caesarean delivery with similar parity, gestation length and age were examined. Muscarinic cholinergic receptors were assayed in frozen sections of the umbilical artery and vein by a radioligand binding assay technique, using [3H]-N-methyl scopolamine (NMS) as a ligand. AChE was demonstrated with a histochemical technique associated with microdensitometry. [3H]-NMS was specifically bound to sections of both umbilical artery and vein in a manner consistent with the labelling of muscarinic cholinergic receptors. The affinity of the radioligand was similar in the two vessels, whereas the maximum density of binding sites (Bmax) was higher in the umbilical vein than in the artery. A faint AChE reactivity was observed in the tunica media of both umbilical artery and vein. In pre-eclampsia, a loss of [3H]-NMS binding sites not accompanied by changes in the affinity of radioligand was found. The decrease of muscarinic cholinergic receptors involved to a greater extent the umbilical artery than the vein. No differences in AChE activity were found at the level of umbilical artery and vein between control and pre-eclamptic subjects. These findings suggest that pre-eclampsia is characterized by a loss of muscarinic cholinergic receptors in the umbilical circulation not accompanied by changes of the acetylcholine catabolizing enzyme AChE. It is possible that the decreased density of vascular muscarinic cholinergic receptors in pregnancy-induced hypertension contribute to the increased resistance of the umbilical circulation occurring in pre-eclampsia.

Radioligand binding assay represents an important technique in pharmacological and pharmaceutical research for assessing the receptor profile of new drugs or of compounds under development. In this study, the pharmacological profile and the receptor specificity of compounds active on dopamine and muscarinic cholinergic receptor subtypes were evaluated using as a receptor source, membrane preparations or frozen sections. Dopamine D1-like receptors were assayed in membrane preparations or frozen sections of rat striatum and kidney with [3H]-SCH 23390 as a ligand. Rat striatum, kidney and atrium were used as a source of dopamine D2-like receptors with [3H]-spiperone as a ligand. The non-selective muscarinic cholinergic receptor antagonist [3H]-N-methyl-scopolamine was used to label muscarinic cholinergic receptors in the rat. Frontal cortex represented the source of M1 receptor subtype, heart the source of M2 receptor subtype, sub maxillary gland the source of M3 receptor subtype and striatum the source of M4 receptor subtype. With the exception of cardiac tissue, no significant differences were noticeable in the affinity of dopaminergic or muscarinic cholinergic compounds tested using membrane particles or 8 microns thick slide-mounted section. In the heart, frozen sections gave lower dissociation constant and inhibition constant values than membranes. The above findings suggest that radioligand binding assay on slide-mounted tissue sections may represent a suitable technique for assessing the receptor profile of drugs under development for the treatment of disorders characterised by dopaminergic or muscarnic cholinergic dysfunction.

Nicardipine is a second generation dihydropyridine-type Ca2+ antagonist with high vascular selectivity and strong cerebral and coronary vasodilatory activity. The compound is used in the treatment of hypertension, primarily in the elderly. In this review the main evidence of the cerebrovascular activity of nicardipine in preclinical studies using in vitro and in vivo models is detailed. A particular physico-chemical property of nicardipine is the almost complete protonation in acid environment. This allows its accumulation in ischemic brain regions and makes it a candidate for the treatment of cerebrovascular disorders characterised by impaired brain perfusion. The main clinical data on the use of nicardipine in cerebral ischemia and related disorders, subarachnoid haemorrhage and stroke, are also reviewed. These studies included 5940 patients affected by chronic cerebrovascular insufficiency (cerebral ischemia, cerebral atherosclerosis mainly associated with hypertension, transient ischemic attacks, sequelae of cerebral infarction, thrombosis or embolia, hypertensive encephalopathy), 1540 patients affected by sequelae of subarachnoid haemorrhage and 206 patients affected by stroke. Both preclinical studies and clinical trials have shown that nicardipine is a safe Ca2+ antagonist with powerful cerebrovascular activity. This suggests its possible use in cerebrovascular disorders in which blockade of Ca2+ channels of the L-type and/or selective cerebral vasodilatation is desirable. Further studies are necessary to establish if modulation of neuronal Ca2+ channels of the L-type by nicardipine may have a neuroprotective effect independent by the cerebrovascular activity of the compound.

The effect of the Ca2+ channel blocker nicardipine on the circle of Willis and the different sized pial arteries was assessed in 20-week-old spontaneously hypertensive rats (SHR) using quantitative image analysis techniques. Normotensive Wistar Kyoto (WKY) rats were also used as a normotensive reference group. In SHR a significant increase of systolic blood pressure (SBP) is noticeable in comparison with WKY rats. The media-to-lumen ratio was increased in the circle of Willis arteries, large sized (diameter > than 150 microns), medium sized (diameter between 150 and 50 microns) and small sized (diameter < than 50 microns), pial artery branches. An increase in the thickness of the tunica media and a luminal narrowing was also seen in medium and small sized pial arteries of SHR in comparison with WKY rats. Treatment with an oral dose of 10 mg/Kg of nicardipine 3 h before the sacrifice significantly reduced SBP in SHR. The drug was without effect on circle of Willis and on large sized pial arteries. Moreover, treatment with nicardipine reduced the thickness of the tunica media, the media-to-lumen ratio and increased the luminal area in medium and small sized pial artery branches. These findings show that treatment of SHR with nicardipine significantly reduces SBP and causes a moderate vasodilatation of arteries regulating cerebrovascular resistance. This property may be useful in avoiding generalized or exaggerated cerebrovascular dilatation which could be accompanied by impaired brain perfusion in hypertension.