L. Annunziato

Silencing or knocking out the Na + /Ca 2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca2+]i levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.

Expression and Function of Somatostatin Receptor Subtype 1 in Human Growth Hormone Secreting Pituitary Tumors Deriving from Patients Partially Responsive or Resistant to Long-Term Treatment with Somatostatin Analogs

The role of somatostatin (SS) receptor subtype 1 (SSTR1) in mediating the inhibitory effect of SS on growth hormone (GH) secreting pituitary tumors has been recently demonstrated. In the present study, we evaluated the effect of the selective SSTR1 agonist BIM-23745 on in vitro GH secretion in GH-secreting pituitary tumor cells, deriving from patients resistant or partially responsive to octreotide long-acting release (octreotide-LAR) or lanreotide therapy in vivo and expressing SSTR1 mRNA. In addition, the inhibiting effect of BIM-23745 on the GH secretion was compared with that of octreotide. Our data demonstrate that (1) SSTR1 receptor was present in 56.25% (9/16) of the GH-secreting adenomas examined; (2) in all GH-secreting pituitary tumors that expressed SSTR1, BIM-23745 significantly inhibited GH secretion in vitro, and (3) when SSTR1 subtype was present in tumors from patients resistant to octreotide-LAR or lanreotide therapy, BIM-23745 was able to inhibit the in vitro GH secretion. In conclusion, the results of the current study suggest that SS analogs selective for the SSTR1 may represent a further useful approach for the treatment of acromegaly in patients resistant or partially responsive to octreotide-LAR or lanreotide treatment in vivo.

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection.

Ischemic preconditioning (IPC), an important endogenous adaptive mechanism of the CNS, renders the brain more tolerant to lethal cerebral ischemia. The molecular mechanisms responsible for the induction and maintenance of ischemic tolerance in the brain are complex and still remain undefined. Considering the increased expression of the two sodium calcium exchanger (NCX) isoforms, NCX1 and NCX3, during cerebral ischemia and the relevance of nitric oxide (NO) in IPC modulation, we investigated whether the activation of the NO/PI3K/Akt pathway induced by IPC could regulate calcium homeostasis through changes in NCX1 and NCX3 expression and activity, thus contributing to ischemic tolerance. To this aim, we set up an in vitro model of IPC by exposing cortical neurons to a 30-min oxygen and glucose deprivation (OGD) followed by 3-h OGD plus reoxygenation. IPC was able to stimulate NCX activity, as revealed by Fura-2AM single-cell microfluorimetry. This effect was mediated by the NO/PI3K/Akt pathway since it was blocked by the following: (a) the NOS inhibitors L-NAME and 7-Nitroindazole, (b) the IP3K/Akt inhibitors LY294002, wortmannin and the Akt-negative dominant, (c) the NCX1 and NCX3 siRNA. Intriguingly, this IPC-mediated upregulation of NCX1 and NCX3 activity may control calcium level within endoplasimc reticulum (ER) and mitochondria, respectively. In fact, IPC-induced NCX1 upregulation produced an increase in ER calcium refilling since this increase was prevented by siNCX1. Moreover, by increasing NCX3 activity, IPC reduced mitochondrial calcium concentration. Accordingly, the inhibition of NCX by CGP37157 reverted this effect, thus suggesting that IPC-induced NCX3-increased activity may improve mitochondrial function during OGD/reoxygenation. Collectively, these results indicate that IPC-induced neuroprotection may occur through the modulation of calcium homeostasis in ER and mitochondria through NO/PI3K/Akt-mediated NCX1 and NCX3 upregulation.

Lack of evidence for an inhibitory role played by tuberoinfundibular dopaminergic neurons on TSH secretion in the rat.

The role of dopamine (DA) in the control of thyroid stimulating hormone (TSH) secretion in basal or cold stimulated conditions was investigated by using pharmacological or neurosurgical tools. The intraventricular injection of DA (5 micrograms/animal) or the subcutaneus (s.c.) injection of a dopaminomimetic agent failed to induce changes of TSH plasma levels in normal or in cold stimulated conditions. The same results were obtained by intraperitoneal (i.p.) administration of haloperidol, a blocker of dopaminergic receptors. The complete deafferentation of hypothalamus, which causes degeneration of norepinephrinergic nerve endings and leaves the DA tuberoinfundibular system unaffected, prevented the TSH release evoked by cold exposure. alpha-Methyl-p-tyrosine (alpha-MpT) (250 mg/kg i.p.), which causes a remarkable reduction of DA in the median eminence (ME) of deafferented animals, was unable to restore the TSH response to cold. Collectively these results seem to suggest that DA does not play a significative role in the control of TSH secretion in the rat.

Chronic treatment with reserpine and adrenocortical activation.

Daily i.p. injection of reserpine for 9 days strongly depletes hypothalamic norephinephrine (NE); after an initial activation, adrenocortical function returns to control values by the 5th day. Tyrosine hydroxylase (TH) activity in the brain stem of reserpine-treated rats exhibits a progressive increase. Alpha-methyl-para-tyrosine (chi-MpT) in rats chronically pretreated with reserpine provokes adrenocortical activation and a further decrease of hypothalamic NE. Exogenous ACTH in the same animals revealed an unimpaired adrenocortical reactivity after prolonged treatment with reserpine. These results seem to suggest that the disappearance of adrenocortical activation following long-term treatment with reserpine is due to the stimulated formation of a small functional pool of NE available for the tonic inhibition of CRF-ACTH secretion.

Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype.

TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily released by microglia, appears to be involved in the induction of apoptosis following focal brain ischemia. Indeed, brain ischemia is associated with progressive enlargement of damaged areas and prominent inflammation. As ischemic preconditioning reduces inflammatory response to brain ischemia and ameliorates brain damage, the purpose of the present study was to evaluate the role of TRAIL and its receptors in stroke and ischemic preconditioning and to propose, by modulating TRAIL pathway, a new therapeutic strategy in stroke. In order to achieve this aim a rat model of harmful focal ischemia, obtained by subjecting animals to 100u2009min of transient occlusion of middle cerebral artery followed by 24u2009h of reperfusion and a rat model of ischemic preconditioning in which the harmful ischemia was preceded by 30u2009mins of tMCAO, which represents the preconditioning protective stimulus, were used. Results show that the neuroprotection elicited by ischemic preconditioning occurs through both upregulation of TRAIL decoy receptors and downregulation of TRAIL itself and of its death receptors. As a counterproof, immunoneutralization of TRAIL in tMCAO animals resulted in significant restraint of tissue damage and in a marked functional recovery. Our data shed new light on the mechanisms that propagate ongoing neuronal damage after ischemia in the adult mammalian brain and provide new molecular targets for therapeutic intervention. Strategies aimed to repress the death-inducing ligands TRAIL, to antagonize the death receptors, or to activate the decoy receptors open new perspectives for the treatment of stroke.

Cytoplasmic alkalinization induced by insulin through an activation of Na(+)-H+ antiporter inhibits tyrosine hydroxylase activity in striatal synaptosomes.

Insulin dose-dependently inhibited tyrosine hydroxylase (TH) activity and increased intrasynaposomal pH (pHi) in rat striatal nerve endings. Both these effects of insulin on TH and pHi were prevented by the 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA), a putative selective inhibitor of the Na(+)-H+ antiporter. Interestingly when, by changing the extracellular pH (pHo), the pHi was increased, from 7.1 up to 7.5, an equivalent inhibition of TH activity occurred. The inhibitory action exerted from insulin on TH activity disappeared when the hormone was added to synaptosomes whose pHi was lowered to 6.83. Collectively, the results of the present study showed that insulin inhibited TH activity in striatal synaptosomes. This effect seems to involve the activation of the Na(+)-H+ antiporter. This exchange system once activated, may induce an intrasynaptosomal alkalinization, a condition in which TH activity is inhibited.

THE ROLE OF CENTRAL NORADRENERGIC AND DOPAMINERGIC NEURONS IN THE CONTROL OF TRH-TSH SECRETION

ABSTRACT The long-lasting depletion of hypothalamic noradrenaline(NE)produced by 6-hydroxydopamine (6-OHDA) prevented the TSH response to cold exposure(CE). In this condition the stimulation of central α-adrenergic receptors with clonidine not only reestablished the response to CE, but also enhanced this effect. The utilization of compounds able to antagonize or stimulate central dopaminergic receptors was unable to modify thyrotropin secretion both in basal and cold-stimulated conditions.

Endogenous dopamine release from tuberoinfundibular neurons: Does calmodulin play any role?

SummaryThe possible involvement of calmodulin in the process of endogenous dopamine (DA) release from arcuateperiventricular nuclei-median eminence fragments, containing tuberoinfundibular dopaminergic (TIDA) neurons, has been investigated in an in vitro incubation system. For this purpose the basal and K+-stimulated DA release was examined in the presence and in the absence of the different putative calmodulin antagonists, pimozide, trifluoperazine, penfluridol and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7).Trifluoperazine and pimozide in concentrations up to 100 μM were both uneffective in blocking K+-evoked DA release. Penfluridol in doses of 5 and 10 μM, did not prevent 35 mM K+-induced endogenous DA release. It was able to reduce K+-stimulated DA release only at the very large concentration of 100 μM.W-7 added in vitro to the hypothalamic fragments, prevented endogenous DA release evoked by 35 mM K+ in a dose-dependent manner. W-5, a chlorine deficient analogue of W-7, that interacts only weakly with calmodulin, failed to modify K+-stimulated endogenous DA release in doses up to 200 μM.All the putative calmodulin antagonists used in the present study did not induce any change of basal DA release.IN conclusion the fact that most of the agents, except W-7, known to antagonize calmodulin-dependent processes in many biological systems failed to interfere with the release of endogenous DA from TIDA neurons seems to suggest that calmodulin does not play a crucial role in the process of DA release and that the inhibitory effect of W-7 on endogenous DA release may be better attributed to other mechanisms different from its anticalmodulin action.

Reduced tuberoinfundibular dopaminergic neuronal function in rats after long-term withdrawal of estrogen treatment

Hypothalamic fragments from female rats treated repeatedly with estradiol valerate (EV) and bearing prolactin (PRL)-secreting tumors contained, seven months after the last EV injection, lower concentrations of dopamine (DA) than age-matched controls. Depolarizing concentrations of K+ (35 mM) and amphetamine (50 μM) evoked in PRL-secreting tumor bearing rats an endogenous DA release significantly lower than in controls.