Induction of abscisic acid-regulated gene expression by diacylglycerol pyrophosphate involves Ca2+ and anion currents in Arabidopsis suspension cells. (1/11)

Diacylglycerol pyrophosphate (DGPP) was recently shown to be a possible intermediate in abscisic acid (ABA) signaling. In this study, reverse transcription-PCR of ABA up-regulated genes was used to evaluate the ability of DGPP to trigger gene expression in Arabidopsis (Arabidopsis thaliana) suspension cells. At5g06760, LTI30, RD29A, and RAB18 were stimulated by ABA and also specifically expressed in DGPP-treated cells. Use of the Ca2+ channel blockers fluspirilene and pimozide and the Ca2+ chelator EGTA showed that Ca2+ was required for ABA induction of DGPP formation. In addition, Ca2+ participated in DGPP induction of gene expression via stimulation of anion currents. Hence, a sequence of Ca2+, DGPP, and anion currents, constituting a core of early ABA-signaling events necessary for gene expression, is proposed.  (+info)

Selective antagonism of calcium channel activators by fluspirilene. (2/11)

1. Fluspirilene has been claimed to bind to a high affinity site in the calcium channel in skeletal muscle. We have investigated its calcium-antagonistic effects in smooth muscle and affinity for the channel in radioligand binding assays. 2. Fluspirilene was weakly active as an antagonist of Ca2(+)-induced contractions in K(+)-depolarized taenia preparations from the guinea-pig caecum, with threshold antagonism starting from concentrations of 30 nM. Nitrendipine, nicardipine and nimodipine were very potent antagonists in this model (threshold antagonism, greater than 1 nM). 3. In contrast, fluspirilene (10-1000 nM) was a potent non-competitive antagonist of the effects of Bay K 8644 (1-3000 nM) on Ca2(+)-induced contractions and, at 10 nM, selectively antagonised the effects of Bay K 8644, abolished the Ca2(+)-channel activator effects of CGP 28392, without changing the calcium antagonist effects of nitrendipine, or modifying the sensitivity of the tissues to Ca2+. In contrast, the dihydropyridines were more effective as antagonists of Ca2+ than of Bay K 8644. Fluspirilene therefore selectively antagonised the effects of dihydropyridine Ca2+ channel activators without affecting the antagonist potency. 4. In radioligand binding experiments, fluspirilene was a potent displacer of [3H]-PN-200-110 binding to rat cerebral cortical membranes (EC50 30 nM), albeit with a low Hill slope (0.66), and was more potent than other lipophilic diphenylalkylamines such as flunarizine and lidoflazine. Fluspirilene interacted non-competitively with [3H]-PN-200-110 and increased dissociation of the radioligand.  (+info)

Blockade of low and high threshold Ca2+ channels by diphenylbutylpiperidine antipsychotics linked to inhibition of prolactin gene expression. (3/11)

The effects of diphenylbutylpiperidine (DPBP) antipsychotics on Ca2+ currents and prolactin (PRL) synthesis were studied in rat pituitary growth hormone (GH) cell lines (GH3 and GH4C1). In whole-cell patch-clamp experiments, DPBPs including fluspirilene, penfluridol, and pimozide at concentrations ranging from 0.25 to 5 microM each blocked current through low threshold T-type as well as high threshold L-type channels. Each of the drugs preferentially blocked T-type current, and complete inhibition was observed at concentrations as low as 1 microM. Inhibition of L-type channels by DPBPS was enhanced at depolarized holding potentials and promoted by prolonged channel activation. At concentrations similar to those which blocked Ca2+ currents, each of the three DPBPs markedly reduced basal PRL production by GH cells. PRL synthesis stimulated by the dihydropyridine Ca2+ agonist R5417 or thyrotropin releasing hormone (TRH) was also inhibited. The inhibitory effects of the DPBPs were observed at the level of gene transcription. Penfluridol and fluspirilene inhibited basal, Ca2(+)- and TRH-stimulated expression of a fusion gene construct containing the 5'-flanking sequence of the rat PRL gene linked to the luciferase gene. The effect was concentration-dependent with the IC50 values for both drugs of less than 1 microM. Nimodipine also reduced basal, R5417, and TRH-stimulated expression of the reporter gene construct. Similar results were obtained with a reporter gene construct containing the 5'-flanking sequence of the rat GH gene. The GH luciferase construct was only slightly responsive to R5417 and TRH; however, these responses were reduced by fluspirilene and nimodipine at concentrations of less than 1 microM. These studies demonstrate that the DPBP antipsychotics block T- as well as L-type Ca2+ channels in GH cells and inhibit PRL production at the level of transcription. They also indicate that functioning Ca2+ channels are necessary for TRH-stimulated PRL gene transcription.  (+info)

Small molecule regulators of autophagy identified by an image-based high-throughput screen. (4/11)

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.  (+info)

Control of basal autophagy by calpain1 mediated cleavage of ATG5. (5/11)

Autophagy functions as an important catabolic mechanism by mediating the turnover of intracellular organelles and protein complexes. Although the induction of autophagy by starvation has been extensively studied, we still understand very little about how autophagy is regulated under normal nutritional conditions. Here we describe a study using a small molecule autophagy inducer, fluspirilene, as a tool to explore the mechanism of autophagy induction in normal living cells. We confirm the activity of fluspirilene in inhibiting Ca(2+) flux. Furthermore, we show that reducing intracellular Ca(2+) prevents the cleavage of ATG5, which in turn increases the levels of full-length ATG5 and ATG12-ATG5 conjugate. Using siRNA mediated gene silencing, we demonstrate that inhibiting calpain1 is sufficient to induce autophagy in living cells. We conclude that calpain1 plays an important role in controlling the levels of autophagy in normal living cells by regulating the levels of a key signaling molecule, ATG12-ATG5 conjugate.  (+info)

Neuroleptics of the diphenylbutylpiperidine series are potent calcium channel inhibitors. (6/11)

[3H]Fluspirilene, a neuroleptic molecule of the diphenylbutylpiperidine series, binds to skeletal muscle transverse tubule membranes with a high affinity corresponding to a Kd of 0.11 +/- 0.04 nM, A 1:1 stoichiometry was found between [3H]fluspirilene binding and the binding of (-)-[3H]desmethoxyverapamil [(-)[3H]D888], one of the most potent Ca2+ channel inhibitors. Ca2+ channel inhibitors such as D888, verapamil, gallopamil, bepridil, or diltiazem antagonize [3H]fluspirilene binding besides antagonizing (-)[3H]-D888 binding. Neuroleptics, especially those of the diphenylbutylpiperidine family, also antagonize both (-)[3H]D888 binding and [3H]fluspirilene binding. There is an excellent correlation between affinities found from [3H]fluspirilene binding experiments and those found from (-)[3H]D888 binding experiments. Analysis of the properties of these cross-inhibitions indicates that [3H]fluspirilene binds to a site that is not identical to that for phenylalkylamine derivatives (gallopamil, verapamil, diltiazem, and bepridil). Voltage-clamp experiments have shown that fluspirilene is an efficient inhibitor of the voltage dependent Ca2+ channel, achieving a half-maximal effect near 0.1-0.2 nM and nearly complete blockade at 1 nM. Fluspirilene blockade has little voltage dependence.  (+info)

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex. (7/11)

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.  (+info)

Visual contrast sensitivity in drug-induced Parkinsonism. (8/11)

The influence of stimulus orientation on contrast sensitivity function was studied in 10 patients with drug-induced Parkinsonism. Nine of the 10 patients had at least one eye with contrast sensitivity deficit for vertical and/or horizontal stimuli. Only generalised contrast sensitivity loss, observed in two eyes, was stimulus orientation independent. All spatial frequency-selective contrast deficits in 15 eyes were orientation dependent. The striking similarity between the pattern of contrast sensitivity loss in drug-induced Parkinsonism and that in idiopathic Parkinson's disease, suggests that generalised dopaminergic deficiency, from whatever cause, affects visual function in an analogous way.  (+info)

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