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Due to the neuroprotective role of the Na+/Ca2+exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1–19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.

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Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting infants caused by alterations of the survival motor neuron gene, which results in progressive degeneration of motor neurons (MNs). Although an effective treatment for SMA patients has been recently developed, the molecular pathway involved in selective MN degeneration has not been yet elucidated. In particular, miR-206 has been demonstrated to play a relevant role in the regeneration of neuromuscular junction in several MN diseases, and particularly it is upregulated in the quadriceps, tibialis anterior, spinal cord, and serum of SMA mice. In the present paper, we demonstrated that miR-206 was transiently upregulated also in the brainstem of the mouse model of SMA, SMAΔ7, in the early phase of the disease paralleling MN degeneration and was down-regulated in the late symptomatic phase. To prevent this downregulation, we intracerebroventricularly injected miR-206 in SMA pups, demonstrating that miR-206 reduced the severity of SMA pathology, slowing down disease progression, increasing survival rate, and improving behavioral performance of mice. Interestingly, exogenous miRNA-206-induced upregulation caused a reduction of the predicted target sodium calcium exchanger isoform 2, NCX2, one of the main regulators of intracellular [Ca2+] and [Na+]. Therefore, we hypothesized that miR-206 might exert part of its neuroprotective effect modulating NCX2 expression in SMA disease.

Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93Atransgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS.

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Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca2+radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC50in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABAAcurrents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor l-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and l-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras—obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2—was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.

The sodium–calcium exchanger-1 (NCX1) represents a key mediator for maintaining Naiand Ca2ihomeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain.

In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor L-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras—obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2—was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.

The sodium–calcium exchanger NCX1 represents a key mediator for maintaining Naiand Ca2iin anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions.

With the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC(50) values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the alpha(2) repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca(2+) channels, tetrodotoxin-sensitive Na(+) channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.

The 3 gene products of the Na/Ca2exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca2refilling in cortical neurons.

The Kv7.2 subunits are the main molecular determinants of the M-current, a widespread K+ current regulating neuronal excitability. Mutations in the Kv7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S4 segment of Kv7.2. In this study, each of the six S4 arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S4 arginine residue plays a relevant role in the voltage-dependent gating of Kv7.2 channels. In particular, a decreased positive charge at the N-terminal end of S4 stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S4 favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.

The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na+and Ca2+ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.

Antithrombin (AT), a glycoprotein belonging to the serpin family, blocks thrombin formation and activity at several steps. Thrombin, beside its relevant role in the coagulation cascade, exerts neurodetrimental effects through the activation of a family of protease-activated receptors, which can be implicated in stroke pathophysiology. The aims of the present study were to evaluate whether AT could reduce brain damage, ameliorate neurologic deficits, and prolong animal survival.

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Naand Ca2ions, which results in subsequent cell death. The plasma membrane Na/Ca2exchanger (NCX), regulates the distribution of these ions acting either in the forward mode or in its reverse mode and it can play a critical role in brain ischemia. However, it is unclear whether the activity of NCX leads to detrimental or beneficial effects.

Nitric oxide (NO) is critical for the normal physiological regulation of the nervous system and other tissues. l-Arginine, but not d-arginine, is the natural substrate for nitric oxide synthase (NOS), for it is enzymatically converted to NO and l-citrulline. However, recent evidence suggests that d-arginine can also produce NO and NO-derivatives via a different pathway. The aim of the present paper was to raise NO levels in the cells by increasing the cell permeation of its precursors. To this aim, two galactosyl prodrugs, l-arginine-d-galactos-6‘-yl ester (l-ArgGal) and d-arginine-d-galactos-6‘-yl ester (d-ArgGal) were synthesized. Remarkably, using the HPLC-ESI/MS technique, we found that l-ArgGal and d-ArgGal prodrugs both increased the concentration levels of l- and d-arginine and their derivatives in pituitary GH3cells. Furthermore, we found that d-ArgGal (1) penetrated cell membranes more rapidly than its precursor d-arginine, (2) released arginine more slowly and in greater amounts than l-ArgGal, and (3) produced much higher levels of DAF-2 monitored NO and nitrite than did l-ArgGal under the same experimental conditions. In conclusion, these results indicate that an increase in the cell permeation of l- and d-arginine by l-ArgGal and d-ArgGal can lead to an increase in NO levels.

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src- and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism.

The present study explores the hypothesis that the new anti-epileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP3)-dependent release of intracellular Ca2+initiated by Gq-coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca2+]iresponse to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca2+]iincrease, elicited either by 1 μM BK or by 100 μM ATP (IC50, 0.39 ± 0.01 μM for BK and 0.20 ± 0.01 μM for ATP; Hill coefficients, 1.33 ± 0.04 for BK and 1.38 ± 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca2+]iresponse to BK, LEV inhibitory effect was mainly exerted on the IP3-dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca2+stored in the intracellular deposits since it did not reduce the amplitude of [Ca2+]iresponse either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca2+release from the IP3-sensitive stores without reducing Ca2+storage into these deposits. Because of the relevant implications of IP3-dependent Ca2+release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

The present study explores the hypothesis that the new anti-epileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP(3))-dependent release of intracellular Ca(2+) initiated by G(q)-coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca(2+)](i) response to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca(2+)](i) increase, elicited either by 1 microM BK or by 100 microM ATP (IC(50), 0.39 +/- 0.01 microM for BK and 0.20 +/- 0.01 microM for ATP; Hill coefficients, 1.33 +/- 0.04 for BK and 1.38 +/- 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca(2+)](i) response to BK, LEV inhibitory effect was mainly exerted on the IP(3)-dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca(2+) stored in the intracellular deposits since it did not reduce the amplitude of [Ca(2+)](i) response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca(2+) release from the IP(3)-sensitive stores without reducing Ca(2+) storage into these deposits. Because of the relevant implications of IP(3)-dependent Ca(2+) release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

Background and Purpose—The NaCa2exchanger, by mediating Ca2and Nafluxes in a bidirectional way across the synaptic plasma membrane, may play a pivotal role in the events leading to anoxic damage. In the brain, there are 3 different genes coding for 3 different proteins: NCX1, NCX2, and NCX3. The aim of this study was to determine whether NCX1, NCX2, and NCX3 might play a differential role in the development of cerebral injury induced by permanent middle cerebral artery occlusion (pMCAO).

In the central nervous system, the NaCa2exchanger plays a fundamental role in controlling changes in the intracellular concentrations of Naand Ca2ions that occur in physiologic conditions such as neurotransmitter release, cell migration and differentiation, gene expression, as well as neuro-degenerative processes. Three genes, NCX1, NCX2, and NCX3, encoding for NaCa2exchanger isoforms have been cloned. In this review, by using non-radioactive in situhybridization and light immunohistochemistry with NCX isoform-specific riboprobes and antibodies, respectively, a systematic brain mapping for both transcripts and proteins encoded by all three NCX genes is described. Intense expression of NCX transcripts and proteins was detected in the cerebral cortex, hippocampus, thalamus, metathalamus, hypothalamus, brainstem, spinal cord, and cerebellum. In these areas, NCX transcripts and proteins were often found with an overlapping distribution pattern, although specific brain areas displaying a peculiar expression of each exchanger isoform were also found. Furthermore, immunoelectron and confocal microscopy revealed the expression of the NCX1 isoform of the exchanger at both pre- and postsynaptic sites as well as in association with membranes of the endoplasmic reticulum. Collectively, these data suggest that the different isoforms of the NaCa2exchanger appear to be selectively expressed in several CNS regions where they might underlie different functional roles.

The K+channels encoded by the human Ether-a-gogo Related Gene-1(hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K+channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopusoocytes. Substitution of the S5-S6linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S5-S6linker generated K+channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K+currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S5-S6linker region of hERG1 K+channels are crucial players in ROS-induced modulation of hERG1 K+channels.

An ever increasing number of reports shows the involvement of free radicals in the functional and structural changes occurring in the brain as a part of the "normal" aging process. Given that plasma membrane and intracellular ion channels play a critical role in maintaining intracellular ion homeostasis, which is crucial for neuronal cell survival, in the present review we have attempted to elaborate on the idea that functional changes in ion channel activity induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) might occur during the aging process. To this aim, we have reviewed the available literature and the data obtained in our laboratory on the ability of ROS and RNS to modify the activity of several plasma membrane and intracellular ion channels and transporters, in an attempt to correlate such changes with those occurring with the aging process. Particular emphasis is given to voltage-gated Na, Ca, and K channels, although second messenger-activated channels like Ca- and ATP-dependent K channels, and intracellular channels controlling intracellular Ca storage and release will also be discussed. On the basis of the available data it is not yet possible to establish a strict correlation between the changes in neuronal electrophysiological properties induced by oxidative modification at the level of ion channels and the neurodegenerative process accompanying brain aging. However, an increasing amount of information suggests that the modulatory effects exerted by ROS and RNS on ion channel proteins might have a relevant role for neuronal cell survival or death. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS and RNS as important mechanisms for the aging process. Only when a more complete molecular picture of the aging process will be available, it will be possible to test the fascinating hypothesis that aging might be pharmacologically delayed by modulating ROS and RNS action on ion channels or the biochemical pathways involved in their modulation.

In the present study, the effects on intracellular calcium concentration ([Ca2+]i) oscillations of the blockade of ether-a-go-go-related gene (ERG) K+channels and of Ca2+influx through store-operated channels (SOC) activated by [Ca2+]istore depletion have been studied in GH3cells by means of a combination of single-cell fura-2 microfluorimetry and whole-cell mode of the patch-clamp technique. Nanomolar concentrations (1–30 nM) of the piperidinic second-generation antihistamines terfenadine and astemizole and of the class III antiarrhythmic methanesulfonanilide dofetilide, by blocking ERG K+channels, increased the frequency and the amplitude of [Ca2+]ioscillations in resting oscillating GH3cells. These compounds also induced the appearance of an oscillatory pattern of [Ca2+]iin a subpopulation of nonoscillating GH3cells. The effects of ERG K+channel blockade on [Ca2+]ioscillations appeared to be due to the activation of L-type Ca2+channels, because they were prevented by 300 nM nimodipine. By contrast, the piperazinic second-generation antihistamine cetirizine (0.01–30 μM), which served as a negative control, failed to affect ERG K+channels and did not interfere with [Ca2+]ioscillations in GH3cells. Interestingly, micromolar concentrations of terfenadine and astemizole (0.3–30 μM), but not of dofetilide (10–100 μM), produced an inhibition of the spontaneous oscillatory pattern of [Ca2+]ichanges. This effect was possibly related to an inhibition of SOC, because these compounds inhibited the increase of [Ca2+]iachieved by extracellular calcium reintroduction after intracellular calcium store depletion with the sarcoplasmic or endoplasmic reticulum calcium ATPase pump inhibitor thapsigargin (10 μM) in an extracellular calcium-free medium. The same inhibitory effect on [Ca2+]ioscillations and SOC was observed with the first-generation antihistamine hydroxyzine (1–30 μM), the more hydrophobic metabolic precursor of cetirizine. Collectively, the results of the present study obtained with compounds that interfere in a different concentration range with ERG K+channels or SOC suggest that 1) ERG K+channels play a relevant role in controlling the oscillatory pattern of [Ca2+]iin resting GH3cells and 2) the inhibition of SOC might induce an opposite effect, i.e., an inhibition of [Ca2+]ioscillations.

Ventricular arrhythmias are rare but life‐threatening side effects of therapy with the second‐generation H1receptor antagonists terfenadine and astemizole. Blockade of the K+channels encoded by the Human Ether‐à‐go‐go‐Related Gene 1 (HERG1) K+channels, which is the molecular basis of the cardiac repolarizing current IKr, by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds.In the present study, the potential blocking ability of the novel second‐generation H1receptor antagonist mizolastine of the HERG1 K+channels heterologously expressed in Xenopusoocytes and in HEK 293 cells or constitutively present in SH‐SY5Y human neuroblastoma cells has been examined and compared to that of astemizole.Mizolastine blocked HERG1 K+channels expressed in Xenopusoocytes with an estimated IC50of 3.4 μM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K+channel were 72.4±11.9 s for 3 μMmizolastine, and 1361±306 s for 1 μMastemizole.In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose‐related inhibitory action on IHERG1, with an IC50of 350±76 nM. Furthermore, mizolastine dose‐dependently inhibited HERG1 K+channels constitutively expressed in SH‐SY5Y human neuroblastoma clonal cells.The results of the present study suggest that the novel second‐generation H1receptor antagonist mizolastine, in concentrations higher than those achieved in vivoduring standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K+channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1‐inhibitory action.

Cabergoline (CAB) (1-[(6-allelylergolin-8β-yl)carbonyl]-1-[3-(dimethylamino)propyl]-3-ethyl-urea) is an ergoline derivative with potent, selective and long-lasting inhibitory activity on prolactin (PRL) secretion acting on dopamine receptors present in pituitary lactotrophes. Receptor binding studies have demonstrated that CAB has high in vitro selectivity and affinity for the subtype D2of the dopamine receptor. In cultures of rat anterior pituitary cells, the concentrations of CAB and bromocriptine required to inhibit PRL secretory activity by 50% (IC50) were 0.1 and 3.4 nmol/l, respectively. As compared to bromocriptine, CAB was more potent in inhibiting the binding of [3H]N-n-propylnorapomorphine and it occupied the receptor for longer. These effects were observed in all areas of the rat brain. In vivo, CAB at doses of 0.125 - 1 mg twice weekly caused a dose-dependent suppression of PRL secretion in women with hyperprolactinaemia. CAB was shown to be significantly more effective than bromocriptine in inducing a complete biochemical response and clinical efficacy and was better tolerated than bromocriptine in the majority of patients. Notable tumour shrinkage until tumour disappearance was observed during CAB treatment in most patients with macroprolactinoma. CAB was also shown to be effective in patients resistant or poorly responsive to bromocriptine. In view of the limited data on CAB-associated pregnancies and the long half-life of the drug, it is currently recommended that women seeking to became pregnant, once ovulatory cycles have been established, should discontinue CAB therapy 1 month before they intend to conceive. However, no data on negative effects on pregnancy or offspring have been reported. The great efficacy of CAB together with its excellent tolerability makes this drug the current treatment of choice for the majority of patients with hyperprolactinaemic disorders. Very recently, the efficacy of CAB treatment has been reported in patients with acromegaly and clinically non-functioning adenomas with controversial results. CAB was also reported to have some efficacy in patients with Nelson’s syndrome and Cushing’s disease although these data are available only for limited case reports.

In the current study, the potential blocking ability of K+channels encoded by the human ether-a-go-go related gene (HERG) by the piperazine H1receptor antagonist cetirizine has been examined and compared with that of other second-generation antihistamines (astemizole, terfenadine, and loratadine). Cetirizine was completely devoid of any inhibitory action on HERG K+channels heterologously expressed inXenopus laevisoocytes in concentrations up to 30 μm. On the other hand, terfenadine and astemizole effectively blocked HERG K+channels with nanomolar affinities (the estimated IC50values were 330 and 480 nm, respectively), whereas loratadine was ∼300-fold less potent (IC50≈ 100 μm). In addition, in contrast to terfenadine, cetirizine did not show use-dependent blockade. In SH-SY5Y cells, a human neuroblastoma clone that constitutively expresses K+currents carried by HERG channels (IHERG), as well as in human embryonic kidney 293 cells stably transfected with HERG cDNA, extracellular perfusion with 3 μmcetirizine did not exert any inhibitory action on IHERG. Astemizole (3 μm), on the other hand, was highly effective. Terfenadine (3 μm) caused a marked (≈80%) inhibition of IHERGin SH-SY5Y cells, whereas loratadine, at the same concentration, caused a 40% blockade. Furthermore, the application of cetirizine (3 μm) on the intracellular side of the membrane of HERG-transfected human embryonic kidney 293 cells did not affect IHERG, whereas the same intracellular concentration of astemizole caused a complete block. The results of the current study suggest that second-generation antihistamines display marked differences in their ability to block HERG K+channels. Cetirizine in particular, which possesses more polar and smaller substituent groups attached to the tertiary amine compared with other antihistamines, lacks HERG-blocking properties, possibly explaining the absence of torsade de pointes ventricular arrhythmias associated with its therapeutical use.

Colao A, Merola B, Ferone D, Calabrese MR, Longobardi S, Spaziante R, Di Renzo G, Annunziato L, Lombardi G. Effect of corticotrophin-releasing hormone administration on growth hormone levels in acromegaly: in vivo and in vitro studies. Eur J Endocrinol 1994;131:14–19. ISSN 0804–4643The ability of CRH to cause a paradoxical response of GH in acromegaly is still under debate. In this study, the effect of CRH administration on GH release was evaluated in a large series of patients with active acromegaly, both in vivo, compared to that of TRH and GnRH, and in vitro. The study was organized as follows. In vivo study: 30 acromegalic patients were submitted to TRH, GnRH, and CRH tests on non-consecutive days: blood samples were collected before and 10, 20, 30, 45, 60, 90, and 120 min after bolus. In nine patients the CRH test was repeated after a 3-month therapy with octreotide and at the dose of 300–600 μg sc thrice daily. In vitro study: CRH (10 nmol/l, 100 nmol/l, and 1 μmol/l) was tested on pituitary tumor tissue obtained in eight patients during transsphenoidal adenomectomy and immediately placed in sterile Ca2+and Mg2+free buffer phosphate. A paradoxical GH response to TRH (evaluated as a GH increase over 50% of basal values) was recorded in 19 patients (63.3%), whereas 7 patients (23.3%) responded to GnRH and 4 others to CRH (13.3%). TRH administration induced a maximal percent GH increase significantly greater than that induced by GnRH and CRH (p < 0.05). Octreotide caused the normalization of GH and insulin-like growth factorI levels in all the patients, as well as the disappearance of the GH paradoxical response to CRH in 3/4 patients. All four CRH responders and four CRH non-responders, used as controls, were surgically treated and adenomatous tissue was used for the in vitro study. No significant GH increase was found in the CRH non-responders and in the three CRH responders in whom the GH paradoxical response to CRH disappeared during octreotide treatment. Conversely, a significant GH increase (over 250% of pre-incubation values) after CRH administration was found in the remaining case. In conclusion, the results of the present study show that CRH is rarely able to stimulate paradoxical GH response in patients with active acromegaly and that the in vivo recorded GH release after CRH is in vitro reproducible only in extremely rare cases.Annamaria Colao, Corso Europa 63, 80127 Napoli, Italy

The objectives of the treatment of hyperprolactinaemia are to suppress excessive hormone secretion and its clinical consequences, to remove tumour mass, to preserve the residual pituitary function and to prevent disease recurrence or progression. Prior to the advent of pharmacotherapy, therapy usually consisted of surgical resection and/or pituitary irradiation. In microprolactinomas, trans-sphenoidal surgical resection normalizes prolactin (PRL) levels, restores normal menses and produces the disappearance of galactorrhoea in a great majority of patients, but normalization of serum PRL levels varies from 35-70%. In macroprolactinomas, trans-sphenoidal surgery is less successful with only 32% of patients appearing to be cured initially. However, the recurrence rate is 19%, and the long-term cure rate is only 26%. In more than 80% of the patients with microprolactinoma, suppression of PRL levels and tumour shrinkage can be achieved with bromocriptine therapy given at doses of 2.5-5 mg per day. In 5-10% of the patients, the appearance of side-effects (nausea, dizziness and postural hypotension) is a limiting factor in continuing the treatment. Dopaminergic compounds cause notable tumour shrinkage in most macroprolactinomas. Treatment with cabergoline, a selective and long-lasting dopamine 2-receptor agonist at weekly doses of 0.5-2 mg has been shown to be effective both in normalizing PRL levels and in inducing tumour shrinkage. Pharmacotherapy with dopamine (DA) agonists is an appropriate first-line treatment for both micro- and macroprolactinomas. Surgery should be recommended for those patients who are severely intolerant of or resistant to DA agonists.

The present study aimed at evaluating the anterior pituitary hormone levels in the inferior petrosal sinuses and in the peripheral blood of 55 patients affected by various pituitary disorders and undergoing perihypophysial phlebography on neurosurgical indication or for diagnostic purposes. The results indicated that in 6 patients with Cushing's disease and in 4 with hyperprolactinemia the secreting adenoma could be localized by inferior petrosal sinus sampling. Furthermore, the concentrations of all the pituitary hormones were found to be higher in the right and/or in the left inferior petrosal sinus than in peripheral blood, showing a clear gradient between central and peripheral samples. Moreover, the evaluation of hormone central/peripheral concentration ratios revealed noteworthy differences, namely, that central/peripheral concentration ratios of GH, ACTH, and PRL were significantly higher than those of TSH, FSH, and LH (p<0.01). On the contrary, no significant differences were found when the concentration ratios of GH, ACTH and PRL or TSH, FSH and LH were compared among themselves. This finding may be attributed to at least two factors: the increased pulsatility and the relatively short biological halftime of polypeptic hormones (GH, ACTH, and PRL) compared with glycoprotein hormones (TSH, FSH, and LH).

In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium‐iodide positive dead cells.Extracellular Na+removal, which activates the Na+‐Ca2+exchanger as a Na+efflux pathway and prevents Na+entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells.During chemical hypoxia, in the presence of extracellular Na+ions, a progressive increase of [Ca2+]ioccurred; in the absence of extracellular Na+ions [Ca2+]iwas enhanced to a greater extent.The blockade of the Na+‐Ca2+exchanger by the amiloride derivative 5‐(N‐4‐chlorobenzyl)‐2′,4′‐dimethylbenzamil (CB‐DMB), lanthanum (La3+) and the Ca2+chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ions on C6 glioma cells exposed to chemical hypoxia.The inhibition of the Na+‐Ca2+antiporter enhanced chemical hypoxia‐induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ions (145 mM), suggesting that the blockade of the Na+‐Ca2+antiporter during chemical hypoxia can lead to increased cell damage.Collectively, these results suggest that activation of the Na+‐Ca2+exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

The aim of this study was to evaluate the effect of protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on Ca2+channels in GH3cells. The activity of Ca2+channels was monitored either by single-cell microfluorometry or by the whole-cell configuration of the patch-clamp technique. Genistein (20-200 μM) and herbimycin A (1-15 μM) inhibited [Ca2+]irise induced either by 55 mM K+or 10 μM Bay K 8644. In addition, genistein and lavendustin A inhibited whole-cell Ba2+currents. By contrast, daidzein, a genistein analogue devoid of PTK inhibitory properties, did not modify Ca2+channel activity. The inhibitory action of genistein on the [Ca2+]iincrease was completely counteracted by the PTP inhibitor vanadate (100 μM). Furthermore, vanadate alone potentiated [Ca2+]iresponse to both 55 mM K+and 10 μM Bay K 8644. The possibility that genistein could decrease the [Ca2+]ielevation by enhancing Ca2+removal from the cytosol seems unlikely since genistein also reduced the increase in fura-2 fluorescence ratio induced by Ba2+, a cation that enters into the cells through Ca2+channels but cannot be pumped out by Ca2+extrusion mechanisms. Finally, in unstimulated GH3cells, genistein caused a decline of [Ca2+]iand the disappearance of [Ca2+]ioscillations, whereas vanadate induced an increase of [Ca2+]iand the appearance of [Ca2+]ioscillations in otherwise non-oscillating cells. The present results suggest that in GH3cells PTK activation causes an increase of L-type Ca2+channel function, whereas PTPs exert an inhibitory role.