Prepuberal intranasal dopamine treatment in an animal model of ADHD ameliorates deficient spatial attention, working memory, amino acid transmitters and synaptic markers in prefrontal cortex, ventral and dorsal striatum.

Intranasal application of dopamine (IN-DA) has been shown to increase motor activity and to release DA in the ventral (VS) and dorsal striatum (DS) of rats. The aim of the present study was to assess the effects of IN-DA treatment on parameters of DA and excitatory amino acid (EAA) function in prepuberal rats of the Naples high-excitability (NHE) line, an animal model for attention-deficit hyperactivity disorder (ADHD) and normal random bred (NRB) controls. NHE and NRB rats were daily administered IN-DA (0.075, 0.15, 0.30 mg/kg) or vehicle for 15 days from postnatal days 28-42 and subsequently tested in the Làt maze and in the Eight-arm radial Olton maze. Soluble and membrane-trapped L-glutamate (L-Glu) and L-aspartate (L-Asp) levels as well as NMDAR1 subunit protein levels were determined after sacrifice in IN-DA- and vehicle-treated NHE and NRB rats in prefrontal cortex (PFc), DS and VS. Moreover, DA transporter (DAT) protein and tyrosine hydroxylase (TH) levels were assessed in PFc, DS, VS and mesencephalon (MES) and in ventral tegmental area (VTA) and substantia nigra, respectively. In NHE rats, IN-DA (0.30 mg/kg) decreased horizontal activity and increased nonselective attention relative to vehicle, whereas the lower dose (0.15 mg/kg) increased selective spatial attention. In NHE rats, basal levels of soluble EAAs were reduced in PFc and DS relative to NRB controls, while membrane-trapped EAAs were elevated in VS. Moreover, basal NMDAR1 subunit protein levels were increased in PFc, DS and VS relative to NRB controls. In addition, DAT protein levels were elevated in PFc and VS relative to NRB controls. IN-DA led to a number of changes of EAA, NMDAR1 subunit protein, TH and DAT protein levels in PFc, DS, VS, MES and VTA, in both NHE and NRB rats with significant differences between lines. Our findings indicate that the NHE rat model of ADHD may be characterized by (1) prefrontal and striatal DAT hyperfunction, indicative of DA hyperactivty, and (2) prefrontal and striatal NMDA receptor hyperfunction indicative of net EAA hyperactivty. IN-DA had ameliorative effects on activity level, attention, and working memory, which are likely to be associated with DA action at inhibitory D2 autoreceptors, leading to a reduction in striatal DA hyperactivity and, possibly, DA action on striatal EAA levels, resulting in a decrease of striatal EAA hyperfunction (with persistence of prefrontal EAA hyperfunction). Previous studies on IN-DA treatment in rodents have indicated antidepressant, anxiolytic and anti-parkinsonian effects in relation to enhanced central DAergic activity. Our present results strengthen the prospects of potential therapeutic applications of intranasal  DA by indicating an enhancement of selective attention and working memory in a deficit model.

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.

Conjunctival instillation of plasminogen eliminates ocular lesion in B6.129P2-Plg(tm1Jld) transgenic mice, a model of ligneous conjunctivitis.

Ligneous conjunctivitis is a severe and rare chronic "idiopathic membraneous" conjunctivitis, characterized by the formation of pseudomembranes mostly on the palpebral surfaces that progressively replace the normal mucosa. Evidence has been provided that ligneous conjunctivitis is caused by a severe systemic plasminogen deficiency with decreased plasminogen antigen and decreased plasminogen functional activities. Objective of the present study is to verify the hypothesis that a topical eye application of plasminogen is able to ameliorate the consequences of this disease. Here we report the results of pre-clinical studies performed to investigate the therapeutic effectiveness of an eye-drop plasminogen preparation in B6.129P2-Plg(tm1Jld) transgenic mice, a model of ligneous conjunctivitis. The entity of protection mediated by plasminogen was evaluated by measuring the extent of the eye lesion by means of a computerized system and dedicated software. The results of the present study clearly showed that the administration for six times a day of plasminogen eye-drop solution in the lesioned eye of animals knock-out for plasminogen gene and developing ligneous conjunctivitis caused a dose and time related reduction of the extent of the ocular lesion. These findings may pave the road for the pharmacological treatment of the ocular lesion associated to the ligneous conjunctivitis that at the present is surgically treated by removing the pseudomembranes generated on the eye.

NO-induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn-SOD activity and expression via Ras/ERK1/2 pathway.

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn-superoxide dismutase (Mn-SOD) expression and activity. In addition, the possible involvement of Ras/extracellular-regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30-min sublethal OGD (95% N(2) and 5% CO(2)). Then, after a 24-h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn-SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L-NAME, a well known NOS inhibitor, the increase in Mn-SOD expression occurring during IPC was reduced and, as a result, IPC-induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn-SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn-SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn-SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post-transcriptionally activates Mn-SOD expression and activity, thus promoting neuroprotection during preconditioning.

Up-regulation and increased activity of KV3.4 channels and their accessory subunit MinK-related peptide 2 induced by amyloid peptide are involved in apoptotic neuronal death.

The aim of the present study was to investigate whether K(V)3.4 channel subunits are involved in neuronal death induced by neurotoxic beta-amyloid peptides (Abeta). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Abeta peptide can up-regulate both the transcription/translation and activity of K(V)3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K(V)3.4 expression and activity can be mediated by the nuclear factor-kappaB (NF-kappaB) family of transcriptional factors; and 3) whether the specific inhibition of K(V)3.4 channel subunit reverts the Abeta peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Abeta(1-42) treatment induced an increase in K(V)3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Abeta peptide caused an increase in I(A) current amplitude carried by K(V)3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-kappaB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K(V)3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Abeta(1-42)-induced increase in K(V)3.4 K(+) currents and to prevent cell death caused by Abeta(1-42) exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K(V)3.4 expression. As a whole, our data indicate that K(V)3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.

ncx1, ncx2, and ncx3 gene product expression and function in neuronal anoxia and brain ischemia.

Over the last few years, although extensive studies have focused on the relevant function played by the sodium-calcium exchanger (NCX) during focal ischemia, a thorough understanding of its role still remains a controversial issue. We explored the consequences of the pharmacological inhibition of this antiporter with conventional pharmacological approach, with the synthetic inhibitory peptide, XIP, or with an antisense strategy on the extent of brain damage induced by the permanent occlusion of middle cerebral artery (pMCAO) in rats. Collectively, the results of these studies suggest that ncx1 and ncx3 genes could be play a major role to limit the severity of ischemic damage probably as they act to dampen [Na+]i and [Ca2+]i overload. This mechanism seems to be normally activated in the ischemic brain as we found a selective upregulation of NCX1 and NCX3 mRNA levels in regions of the brain surviving to an ischemic insult. Despite this transcript increase, NCX1, NCX2, and NCX3 proteins undergo an extensive proteolytic degradation in the ipsilateral cerebral hemisphere. All together these results suggest that a rescue program centered on an increase NCX function and expression could halt the progression of the ischemic damage. On the basis of this evidence we directed our attention to the understanding of the transductional and transcriptional pathways responsible for NCX upregulation. To this aim, we are studying whether the brain isoform of Akt, Akt1, which is a downstream effector of neurotrophic factors, such as NGF can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of ncx1, ncx2, and ncx3 gene products.

HIF-1alpha reveals a binding activity to the promoter of iNOS gene after permanent middle cerebral artery occlusion.

Hypoxia inducible factor (HIF-1)-1alpha is a specific, oxygen-sensitive protein that regulates the activity of HIF-1, a transcriptional factor that increases after cerebral ischemia and may either promote or prevent neuronal survival. In this study to determine whether the inducible nitric oxide synthase (iNOS) gene containing the sequence of the hypoxia-responsive enhancer (HRE) was an HIF-1 target after cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO), electrophoretic mobility shift assay (EMSA) and iNOS western blot analysis were performed in the ischemic core, in the area surrounding the infarct and in the hippocampus ipsilateral and contralateral to the lesion. In addition, both HIF-1alpha mRNA and protein expression were examined in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the insult. Our results revealed that pMCAO up-regulates iNOS protein in the ischemic core, in the area surrounding the ischemic core and in the hippocampus ipsilateral to the lesion, and that the activation of iNOS expression is mediated by HIF-1. Moreover, HIF-1alpha mRNA and protein levels increased in the ischemic core and in the hippocampus ipsilateral to the lesion compared with the levels obtained in the corresponding areas of sham-operated controls or in the contralateral hemisphere. Particularly in the area surrounding the ischemic core, HIF-1alpha protein accumulated during pMCAO although mRNA did not increase. Our study suggests that the activation of HIF-1 might be involved in the mechanisms whereby iNOS promotes cell survival and/or death after cerebral ischemia.

Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase.

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.

Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation.

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N(2) and 5% CO2 in a glucose-free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase.

Intracellular signalling mediating HIV-1 gp120 neurotoxicity.

During the last few years several studies have been undertaken to characterise the role of gp120, the HIV-1 envelope glycoprotein, in the pathogenesis of neurological defects associated with AIDS. However, neurons did not appear to be the main target of the virus, since the widespread neuronal damage is not associated with a productive viral infection in neurons. The current opinion supports the hypothesis that an indirect mechanism exists to explain the neuronal cell death which occurs in patients infected by HIV-1. In particular, several reports suggest that gp120 may be the main candidate as mediator of the neurological deficits during HIV-1 infection and demonstrate that this molecule affects neuronal survival through a direct interaction with non-neuronal cell types such as monocytes, macrophages/microglia and astrocytes.

Oncogene transformation of PC Cl3 clonal thyroid cell line induces an autonomous pattern of proliferation that correlates with a loss of basal and stimulated phosphotyrosine phosphatase activity.

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTP eta, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTP mu, was unchanged. Thus, PTP eta may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.

TGF-beta1 prevents gp120-induced impairment of Ca2+ homeostasis and rescues cortical neurons from apoptotic death.

HIV-1 infection frequently induces neuronal death responsible for the development of neurological deficits associated with AIDS. Several reports suggest that gp120, the HIV-1 envelope glycoprotein, is the main candidate as mediator of the HIV-1-dependent neurotoxicity. Here we report the effect of gp120 on the survival of cortical neurons in vitro and the possible mechanisms whereby it occurs. Mature cortical neurons, cultured on a feeder layer of astrocytes, were treated with gp120 in a defined culture medium in absence of serum. The treatment with gp120 induced time-dependent neuronal damage displaying apoptotic features, as revealed by in situ labelling of DNA fragmentation. TGF-beta1, a cytokine that has been previously shown to exert neuroprotective effects, prevented the cell death induced by exposure of cortical neurons to gp120. The prolonged treatment with gp120 also increased neuronal [Ca2+]i, while the coincubation with TGF-beta1 completely prevented the impairment of neuronal Ca2+ homeostasis. These data, taken together, demonstrate that gp120 induces apoptosis in cortical neurons, an effect that can be related to the impairment of Ca2+ homeostasis, and that TGF-beta1 pretreatment reverts both the neuronal death and the alterations in neuronal [Ca2+]i.

Acetyl-L-carnitine arginine amide prevents beta 25-35-induced neurotoxicity in cerebellar granule cells.

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with beta 25-35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KCl in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later beta-amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with beta 25-35 was able to rescue neurons from beta 25-35-induced neurotoxicity. We also studied the changes in Ca2+ homeostasis following glutamate stimulation, in control and beta-amyloid treated single cells, either in presence or in absence of ST857. beta 25-35 did not affect basal [Ca2+]i, while modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase of [Ca2+]i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with beta 25-35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca2+ homeostasis.

Intracellular calcium rise through L-type calcium channels, as molecular mechanism for prion protein fragment 106-126-induced astroglial proliferation.

The infectious prion protein (PrPSc) is the etiologic agent of transmissible neurodegenerative conditions such as scrapie or Creutzfeldt-Jakob disease. Its fragment 106-126 (PrP106-126) has been reported to maintain most of the pathological features of PrPSc. We report here the intracellular mechanisms mediating the proliferative effects of PrP106-126 on rat cortical type I astrocytes. The proliferative effects of PrP106-126 started after 24h of treatment and lasted up to 9 days and was antagonized by the L-type voltage-sensitive calcium channel blocker nicardipine. Microfluorimetric studies showed that PrP106-126 caused a rapid increase in the [Ca+2]i. This effect was prevented by nicardipine, or by Ca(+2)-free conditions, showing that the PrP106-126 enhances [Ca+2]i mobilizing Ca+2 from the extracellular environment. Moreover, binding studies demonstrated a direct interference of PrP106-126 with the dihydropyridine binding site. This is the first evidence that a prion protein fragment directly stimulates the proliferation of astrocytes via an increase in [Ca+2]i through the L-type voltage-sensitive calcium channels.

beta 25-35 alters calcium homeostasis and induces neurotoxicity in cerebellar granule cells.

We studied the neurotoxic effects of beta 25-35 amyloid fragment (beta 25-35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 mM KCI but slightly modified the survival of 25 mM KCI-cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3-DIV-treated cultures; whereas in beta 25-35-pretreated cells, a significant glutamate toxicity was observed. Treatment of 6-DIV cells with beta 25-35, performed with 25 mM KCI, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate-related neurotoxicity, and this effect was enhanced by beta 25-35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of beta 25-35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and beta 25-35-treated cells. beta 25-35 did not affect basal [Ca2+]i but modified glutamate-induced [Ca2+]i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that beta 25-35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.

Absence of D2S dopamine receptor in the prolactin-secreting MMQ pituitary clone: characterization of a wild D2L receptor coupled to native transduction mechanisms.

We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.

Alpha 1 B, but not alpha 1A, adrenoreceptor activates calcium influx through the stimulation of a tyrosine kinase/phosphotyrosine phosphatase pathway, following noradrenaline-induced emptying of IP3 sensitive calcium stores, in PC Cl3 rat thyroid cell line.

In PC Cl3 rat thyroid cell line noradrenaline-induced Ca2+ response, mainly due to the activation of alpha 1B receptors, is characterized by a rapid peak phase, due to the Ca2+ mobilization from inositol trisphosphate-sensitive internal stores, followed by a sustained plateau, representing the capacitative calcium entry. The plateau phase elicited by noradrenaline returns to the basal value within 100 sec from the removal of agonist. The tyrosine kinases inhibitor genistein completely abolishes the plateau upon noradrenaline withdrawal. On the contrary, the tyrosine phosphatases blocker, vanadate, potentiates the plateau phase of calcium response to noradrenaline and prevents the gradual decrease of [Ca2+]i after removal of noradrenaline. The noradrenaline-induced Ca2+ influx, due to the activation of alpha 1A receptor-operated Ca2+ entry is not affected by vanadate. The treatment with noradrenaline induced the tyrosine phosphorylation of specific substrates in lysates derived from PC Cl3 cells, an effect inhibited by genistein pretreatment. These results show that a balance between tyrosine phosphorylation and dephosphorylation is required for the regulation of capacitative calcium entry following noradrenaline stimulation of alpha 1B receptor, whilst the influx of Ca2+ directly operated by alpha 1A receptor activation seems to be independent of the tyrosine phosphorylating pathway.

Apoptosis in cerebellar granule cells is blocked by high KCl, forskolin, and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis.

Cerebellar granule cells deprived of depolarizing concentration of extracellular potassium, [K+]o, undergo apoptosis. We here report that this apoptotic process is associated with an immediate and permanent decrease in the levels of free intracellular calcium, [Ca2+]i. Although forskolin and IGF-1 are both able to prevent apoptosis, only forskolin is able to counteract the instantaneous decrease of [Ca2+]i. However, the early effect of forskolin on [Ca2+]i is lost after longer incubation in low [K+]o. The calcium antagonist nifedipine is able to inhibit the survival effect of high [K+]o, while not affecting forskolin and IGF-1 promoted survival, as assessed by viability and genomic DNA analysis. Accordingly, the L-type calcium channels agonist Bay K8644 significantly enhanced the survival of low KCl treated neurons. To temporally characterize the signal transduction events and the essential transcriptional step in cerebellar granule cells apoptosis, we determined the time course of the rescue capacity of high [K+]o, forskolin, IGF-1, and actinomycin D. Addition of high KCl, forskolin, or IGF-1 6 hr after the initial KCl deprivation saves 50% of cells. Remarkably, 50% of neurons loss the potential to be rescued by actinomycin D after only 1 hr in low [K+]o. Finally, we show that the survival promoting activities of high [K+]o, forskolin, and IGF-1 do not require RNA synthesis. We conclude that [Ca2+]i is involved in the survival promoting activity exerted by high [K+]o but not in those of forskolin and IGF-1, and that all three agents, although rescuing neurons from apoptosis through distinct mechanisms of action, do not necessitate RNA transcription.

Alpha 1A- and alpha 1B-adrenergic receptors mediate the effect of norepinephrine on cytosolic calcium levels in rat PC C13 thyroid cells: thyrotropin modulation of alpha 1B-linked response via a adenosine 3',5'-monophosphate-protein kinase-A-dependent pathway.

The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC C13 rat thyroid cells and identify the molecular mechanisms by which TSH regulates the noradrenergic response. We studied TSH regulation of norepinephrine-induced cytosolic calcium increase by means of the fluorescent probe fura-2. In PC C13 cells grown and maintained in a medium containing TSH (PC C13 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC C13 starved from TSH 5 days before the experiments (PC C13 5H). In both group of cells the calcium response to norepinephrine was concentration dependent and reduced by the removal of extracellular calcium ions. Reintroduction of TSH in the culture medium of the PC C13 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC C13 6H. This effect was complete after a 48-h incubation period and was abolished by the simultaneous treatment of the cells with the protein synthesis inhibitor cycloheximide, suggesting that TSH may stimulate the synthesis of alpha 1-adrenergic receptors in PC C13 cells. Because in these cells we found that TSH increased cAMP levels as well as inositol phosphate production, we tested whether the activation of a protein kinase-A and/or protein kinase-C was involved in TSH regulation of the adrenergic response. We found that the treatment of PC C13 5H cells with forskolin restored the effect of norepinephrine on the calcium level, and that KT5720, an inhibitor of the protein kinase-A, was able to prevent the recovery of the noradrenergic response induced by the readdition of TSH to the culture medium of PC C13 5H. Conversely, treatment of PC C13 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC C13 6H and, to a lesser extent, in PC C13 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize alpha 1-adrenergic receptor subtypes mediating the effects of norepinephrine in PC C13 cells, we used antagonists of alpha 1A and alpha 1B receptors (WB4101 and chlorethylclonidine respectively).(ABSTRACT TRUNCATED AT 400 WORDS)