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We investigated the role of the HIV-1 protein Tat in AIDS-associated dementia, by studying its toxicity on rat cortical and hippocampal neurons in vitro. We evaluated the involvement of astroglial cells and of caspase transduction pathway in determining Tat toxicity. Here we report that synthetic Tat(1-86) induced apoptotic death on cultured rat neurons in a time-dependent manner that was not influenced by glial coculture, and that was abolished by blocking caspase transduction pathway. A microfluorimetric analysis on the Tat excitatory properties on neurons, and its effect on intracellular calcium concentrations, revealed that Tat(1-86) induced increase in cytoplasmic free calcium concentrations in rat hippocampal and cortical neurons. This effect required extracellular calcium and was differently reduced by voltage dependent calcium channel blockers and both NMDA and non-NMDA glutamate receptors antagonists. Furthermore, we observed that Tat(1-86)-treated neurons showed increased sensitivity to the glutamate excitotoxicity. Thus, the Tat-induced neuronal injury seems to occur through a direct interaction of the protein with neurons, requires activation of caspases, and is likely to derive from Tat(1-86)-induced calcium loads and disruption of glutamatergic transmission., (Copyright 2001 Academic Press.)

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).

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.

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.

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.

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.

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)

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin.

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.

Recent findings indicate that interleukin-1 beta (IL1 beta), a monokine secreted by stimulated macrophages and monocytes, modulates neuroendocrine functions in a manner similar to classical hormones. In this study we show that IL1 modulates PRL secretion, assessed by reverse hemolytic plaque assay, and describe the effect of the monokine on adenylate cyclase activity and calcium fluxes in rat normal pituitary cells. In basal and vasoactive intestinal peptide (VIP)-stimulated conditions, low doses of IL1 reduced the mean plaque area, a direct index of PRL secretion without affecting the percentage of PRL-secreting cells. Similarly, low concentrations of IL1 inhibited adenylate cyclase activity in both basal and VIP-stimulated conditions, while higher concentrations restored the enzymatic activity to the control value. IL1 also caused a biphasic effect on the free intracellular calcium increase induced by maitotoxin, a calcium channel activator, being inhibitory at low and stimulatory at high concentrations. The effects of IL1 on adenylate cyclase activity and calcium fluxes were reversed by preincubation of the monokine with its polyclonal antibody, thus confirming the specificity of the effects. In conclusion, our data show that IL1 modulates PRL secretion by acting directly on pituitary cells through interaction with the adenylate cyclase-cAMP system and calcium flux.

Calcium (Ca2+) ionophore A23187 increased the intracellular cAMP content and PRL release in normal rat anterior pituitary cells. Cotreatment with dopamine reduced both control and A23187-stimulated cAMP accumulation and PRL release. The dopamine antagonist spiperone restored the response of cAMP to ionophoric stimulation after pretreatment with dopamine in the greatest concentration used. Penfluridol, a compound with Ca2+-calmodulin-blocking properties, decreased control and A23187-stimulated cAMP content and PRL release. W7, a selective calmodulin-blocking agent, reduced basal cAMP and PRL release, whereas W5, a W7 analog with only 20% of its calmodulin-blocking ability, did not affect cAMP or PRL secretion. These data indicate that the Ca2+-calmodulin and cAMP systems are interrelated in the regulation of PRL secretion. They are also consistent with the hypothesis that the inhibition of PRL release by dopamine occurs after Ca2+ is mobilized and when or before it stimulates adenylate cyclase activity.

In the present study we used 235-1 cells, a prolactin secreting clone derived from a pituitary tumor. In these cells maitotoxin, a calcium channels activator, likely acting on voltage sensitive calcium channels, increases intracellular free calcium measured by Quin 2 technique. Maitotoxin stimulation of calcium flux was inhibited both by nicardipine and verapamil in a dose dependent manner. Pertussis toxin pretreatment does not modify maitotoxin activation of calcium channels, while completely abolishes nicardipine inhibition of maitotoxin induced voltage sensitive calcium channels activation, without affecting verapamil effect. These results suggest a possible involvement of a pertussis toxin sensitive G protein in dihydropyridine inhibition of voltage sensitive calcium channels.

In the present study we investigated the effect of the interleukin 1 beta on intracellular free calcium concentrations in 235-1 cell line both in basal conditions and after stimulation by the calcium channel activator maitotoxin. Interleukin 1 beta (from 0.01 pM to 10 nM) was unable to significantly affect basal cytosolic free calcium levels in acute conditions. The preincubation of these cells with interleukin 1 beta for 48h modulates maitotoxin stimulation of calcium fluxes without modifying basal intracellular free calcium levels. Low concentrations of interleukin 1 beta (0.01 pM, 1 pM) caused a marked reduction of intracellular free calcium concentrations increase induced by maitotoxin while higher doses of the monokine potentiated maitotoxin stimulation of calcium fluxes. The specificity of interleukin 1 beta effect was tested by means of polyclonal anti-interleukin 1 beta antibody (titer 1:100) which significantly abolished the inhibitory effect of interleukin 1 beta on free cytosolic calcium levels. These results show that a long lasting interaction of interleukin 1 beta with its receptor is able to influence voltage-sensitive calcium channels activation induced by maitotoxin in 235-1 cells.

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.

The dopaminergic inhibition of anterior pituitary adenylate cyclase activity, cAMP accumulation, and prolactin release was studied in the presence of the Ca2+ channel activator, maitotoxin. In isobutylmethylxanthine (IBMX)-treated cells, maitotoxin stimulated prolactin secretion within 30 s and cAMP accumulation within 1 min. Although dopamine reduced cAMP accumulation and prolactin release, the effectiveness of the catecholamine was reduced in the presence of maitotoxin. When hemipituitary glands were exposed for 10 min to 100 ng maitotoxin/ml, their membranes showed increased adenylate cyclase activity. The hypothesis that maitotoxin stimulates adenylate cyclase activity by increasing Ca2+ availability was supported by the observation that, at concentrations up to 100 microM, Ca2+-stimulated anterior pituitary adenylate cyclase activity. Although dopamine decreased basal and maitotoxin-stimulated pituitary cAMP accumulation, via changes in adenylate cyclase activity, the decrement in cyclic nucleotide production, but not prolactin release, can be ascribed to the effect of the catecholamine on the basal activities of these parameters. These data provide additional evidence that an increased Ca2+ flux is stimulating to cAMP generation and prolactin release, whereas dopamine is inhibitory to these processes.

The 235-1 pituitary tumor clone was utilized to study prolactin secretion after perturbing cyclic AMP and calcium metabolism. Cellular cyclic AMP levels were elevated after treatment with PGE1, cholera toxin, forskolin, isobutylmethylxanthine as well as dibutryl cyclic AMP; these cyclic AMP responses were associated with increased prolactin release. Ionophore A23187 and maitotoxin, which enhance calcium uptake into cells, also amplified prolactin secretion. In contrast, the calmodulin antagonists penfluridol and W7 reduced basal prolactin release. These data support the hypothesis that cyclic AMP, calcium and calmodulin can participate in prolactin release from 235-1 cells.

The rat pituitary tumor 7315a secretes PRL and ACTH. Although dopamine has no effect on unstimulated PRL release from this tumor, dopamine decreases the adenylate cyclase activity in tumor cell homogenates in a manner similar to that in normal pituitary tissue. However, it was observed that under basal conditions, 7315a tumor cells have an abnormal calcium metabolism because 1) basal PRL release from tumor cells is not modified by the calcium channel blocker D-600 and is only moderately decreased by low calcium, treatments that markedly decrease normal pituitary PRL release; 2) D-600 had no effect on basal 7315a tumor calcium uptake, but blocked the increase in calcium uptake due to the calcium channel activator maitotoxin; 3) increasing the medium Ca+2 concentration above 5 mM increases 7315a PRL release, whereas this treatment decreases PRL release from normal pituitary cells. Maitotoxin and the calcium ionophore A23187 increased 7315a tumor cell PRL release in a manner similar to that in normal pituitary cells. Because dopamine blocks PRL release induced by maitotoxin, A23187, or elevated medium calcium concentration in 7315a tumor cells, the refractoriness of basal 7315a tumor cell PRL release to dopamine may be due to the abnormal calcium balance of the tumor cells under basal conditions.

The marine dinoflagellate toxin maitotoxin (MTX), an activator of calcium channels, stimulates the release of prolactin (PRL), growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) from monolayers of anterior pituitary cells in a dose-dependent manner. Maitotoxin significantly increased PRL, GH, and LH release within 1.5 min and TSH release within 3.5 min, and the stimulation continued for at least 1 h (P less than 0.01). MTX-stimulated hormonal release was blocked by the calcium channel blocker manganese (P less than 0.01). In freshly dispersed perifused pituitary cells in columns, exposure to MTX for 10 min markedly increased PRL, GH, TSH, and LH release for at least 1 h after withdrawal of the toxin. In other experiments, MTX significantly stimulated 45Ca2+ exchange by dispersed pituitary cells within 30 s, continuing for at least 30 min. We conclude that MTX increases anterior pituitary hormonal release, possibly by activating calcium channels, thereby increasing cellular calcium influx. Thus MTX may be a useful agent for investigating the involvement of Ca2+ in hormonal secretory processes.