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Biomedical subjects

E Claro

Publications and source records attributed to E Claro.

At least 19 recordsLinked to original sources
PubMed2001

The cholecystokinin analogues JMV-180 and CCK-8 stimulate phospholipase C through the same binding site of CCK(A) receptor in rat pancreatic acini.

1. This study was designed to address the controversy related to the involvement of phospholipase C in the signalling pathway linked to CCK(A) receptor stimulation by the cholecystokinin analogue JMV-180, a full agonist for amylase release, in rat pancreatic acini. 2. JMV-180 was shown to stimulate phospholipase C by measuring the incorporation of [(32)P]-orthophosphoric acid ([(32)P]-Pi) into phosphatidic acid (PtdOH) and phosphatidylinositol (PtdIns). Both responses elicited by JMV-180 were time and concentration dependent. Maximal effects elicited by JMV-180 were 39.08+/-0.72 and 8.02+/-0.40% for the labelling of [(32)P]-PtdIns and [(32)P]-PtdOH, respectively, as compared to the maximal effects of CCK-8, a full agonist of the CCK(A) receptor. 3. [(32)P]-Pi incorporation into PtdOH and PtdIns was sensitive to lithium, demonstrating that both responses are a consequence of phospholipase C activation. However, since lithium blocks the phosphoinositide cycle by an uncompetitive mechanism, its effect was only apparent at high concentrations of CCK-8 (>10 pM), which elicited stimuli above 20 and 60% of the maximal [(32)P]-PtdOH and [(32)P]-PtdIns labelling, respectively. 4. JMV-180 inhibited the incorporation of [(32)P]-Pi into PtdOH and PtdIns as stimulated by CCK-8, down to its own maximal effect. The estimated IC(50) values for the inhibition curves were not significantly different from those calculated assuming the same single binding site for both agonists. These results indicated that the well established role of JMV-180 as a partial agonist for CCK(A) receptor-linked signalling responses, also applies for the stimulation of phospholipase C. 5. The comparison of CCK-8 and JMV-180 dose-response curves of amylase release to those of PtdIns and PtdOH labelling with [(32)P]-Pi showed the existence of an amplification mechanism between phospholipase C and amylase release for both agonists. 6. In conclusion, we show that JMV-180, as well as CCK-8, stimulate phospholipase C upon interaction with the same binding site at the CCK(A) receptor in rat pancreatic acini.

Amylases↗
PubMed2000

Cholecystokinin octapeptide CCK-8 and carbachol reduce [(32)P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini.

We have studied phospholipase D activation in [(32)P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of (32)P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [(32)P]phosphatidylethanol and [(32)P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of (32)P-phosphatidylalcohol, yet they decreased [(32)P]phosphatidylcholine content and stimulated the generation of [(32)P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity.

Animals↗
PubMed2000

Inhibition of phosphatidylcholine synthesis precedes apoptosis induced by C2-ceramide: protection by exogenous phosphatidylcholine.

Cerebellar granule neurons in primary culture underwent apoptosis when exposed to C2-ceramide. Addition of exogenous phosphatidylcholine (PtdCho) resulted in a dose-dependent full prevention of neuronal death. Exogenous PtdCho also prevented apoptosis induced by farnesol, N-oleoylethanolamine, and sphingomyelinase, but did not prevent apoptosis induced after lowering the potassium concentration in the medium to non-depolarizing levels. Moreover, C2-ceramide inhibited labeling of [32P]PtdCho in cells incubated with [32P]orthophosphate, with the same potency to that causing apoptosis. Although cell viability did not decrease during the first few hours, inhibition of PtdCho synthesis was already patent after a 1 h exposure to C2-ceramide. Taken together, these results strongly suggest that inhibition of PtdCho synthesis constitutes one of the primary events by which C2-ceramide triggers apoptosis in cerebellar granule neurons.

Animals↗
PubMed1999

Fluoride-induced depletion of polyphosphoinositides in rat brain cortical slices: a rationale for the inhibitory effects on phospholipase C.

Fluoride, which is used commonly as a pharmacological tool to activate phosphoinositide-phospholipase C coupled to the heterotrymeric Gq/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P2) in membranes from rat brain cortex. Fluoride enhanced basal production of 3H-inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [3H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPgammaS. However in both cases fluoride depleted [3H]PtdIns4P content by 95%. The inhibitory effects of fluoride on the release of 3H-inositol phosphates in slices were not apparent in a pulse [3H]inositol-labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [3H]inositol in the presence of fluoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3H-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to fluoride poisoning, unaccessibility of the [3H]inositol label to phospholipase C or desensitization of Gq/11, as the effect of carbachol and GTPgammaS was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of fluoride. In conclusion, our data show that fluoride, at a concentration similar to that used to stimulate directly Gq/11-coupled phospholipase C, effectively blocks the synthesis of phospholipase C substrates from PtdIns.

Animals↗
PubMed1998

Partial lithium-associated protection against apoptosis induced by C2-ceramide in cerebellar granule neurons.

Primary cultures of cerebellar granule neurons, maintained in a serum-containing medium, underwent apoptosis when exposed to C2-ceramide, as assessed by mitochondrial reduction of MTT and intranucleosomal DNA fragmentation. After an 18 h exposure to 50 microM C2-ceramide, cell viability decreased by 25-40%. Addition of lithium together with C2-ceramide resulted in a partial protection of apoptosis, which was maximal at 5 mM lithium (37% protection). When lithium was added 5 h before the apoptotic stimulus the neuroprotective effect of the ion was clearly increased (66% protection). This effect was not due to intracellular inositol depletion or inhibition of NMDA receptors. Our data broaden the nature of apoptotic insults being reversed by lithium, stressing the neuroprotective effects of the ion.

Animals↗
PubMed1996

Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices.

Noradrenaline- and ionomycin-stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H-fatty acids showed that two different classes of phosphatidylcholine, separated by two-dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P-phospholipids extracted from [32P]Pi-labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.

Animals↗
PubMed1995

Histamine H1 and endothelin ETB receptors mediate phospholipase D stimulation in rat brain hippocampal slices.

Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans-(1S,3R)-aminocyclopentyl-1,3-dicarboxylic acid (trans-ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans-ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 microM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 nM) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

Animals↗
PubMed1995

Neurotransmitter-specific profiles of inositol phosphates in rat brain cortex: relation to the mode of receptor activation of phosphoinositide phospholipase C.

Phosphoinositide breakdown, as stimulated by six different neurotransmitter receptor agonists (carbachol, serotonin, norepinephrine, trans-(+/-)-aminocyclopentyl-1,3-dicarboxylic acid, endothelin-1 and histamine), has been studied in rat brain cortical slices. The accumulation was monitored of total 3H-inositol phosphates (InsPs) and [3H]CDP-diacylglycerol (CDP-DAG) in [3H]inositol or [3H]cytidine-prelabeled tissue, respectively, and the profile of the major InsPs was quantified as the index log [(inositol 4-monophosphate + inositol 1,4-bisphosphate)/inositol 1-monophosphate]. The efficacy of the six agonists to stimulate the accumulation of CDP-DAG, relative to that of InsPs, was not constant, which revealed varying degrees of defective recycling of DAG to CDP-DAG. The value of the index for the profile of InsPs was not constant either but was characteristic of each agonist. Both parameters (ratio of efficacies CDP-DAG/InsPs and InsPs profile) were not independent and defined two groups of agonists as follows: group a, carbachol and serotonin, with balanced CDP-DAG and InsPs responses, and Ins1P prevailing against inositol 4-monophosphate + inositol 1,4-bisphosphate and group b, norepinephrine, trans-(+/-)-aminocyclopentyl-1,3-dicarboxylic acid, endothelin-1 and histamine, with weak CDP-DAG responses and high accumulation of inositol 4-monophosphate + inositol 1,4-bisphosphate compared with that of inositol 1-monophosphate. In a membrane preparation from brain cortex, only agonists in group a stimulated phospholipase C in the presence of guanosine 5'-O-(3-thiotriphosphate) and in a receptor antagonist-sensitive fashion, which indicated that brain cortical alpha-1, H1, endothelin and glutamate metabotropic receptors stimulate phospholipase C indirectly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Neurotransmitter-stimulated breakdown of endogenous polyphosphoinositides in post mortem human brain.

Membranes from human brain cortex (8-12 h post mortem) were labelled with [3H]inositol, in the presence of CMP, through the back reaction catalysed by PtdIns synthase. The enzyme incorporated [3H]inositol into phosphoinositides at a maximal rate of 419 pmol min-1 mg protein-1. In the absence of CMP, the labelling rate due to the PtdIns headgroup exchanging enzyme was 36 pmol min-1 mg protein-1. Human brain PtdIns synthase showed Kmapp values of 0.49 mM and 18 microM for inositol and CMP, respectively. In the presence of ATP, [3H]polyphosphoinositides formed after [3H]PtdIns were hydrolysed by phospholipase C in a GTP gamma S and neurotransmitter receptor agonist-dependent manner. Production of 3H-inositol phosphates as stimulated by GTP gamma S (350% of basal) was increased by the muscarinic agonists carbachol and oxotremorine-M (600% of basal) and by serotonin (485% of basal). The relative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling between muscarinic and serotonin receptors and phospholipase C is preserved in membranes from post mortem human brain cortex and validate the use of a method involving direct [3H]inositol labelling of a membrane fraction to study the functional state of phospholipase C-coupled receptors in human brain samples.

Aged↗
PubMed1993

Endogenous phosphoinositide precursors of inositol phosphates in rat brain cortical membranes.

The appearance of Ins1P as an index of direct PtdIns breakdown by phospholipase C was examined in rat brain cortical membranes using either exogenous [3H]PtdIns substrate or [3H]inositol-prelabeled endogenous phosphoinositide substrates. Production of [3H]Ins1P was observed using exogenous [3H]PtdIns but not with endogenous substrate over the physiological range of calcium concentrations. [3H]Ins1,4P2 and [3H]Ins4P, derived from phospholipase C breakdown of polyphosphoinositides, were formed by membranes from both exogenous [3H]PtdIns and endogenous 3H-phosphoinositides, in the presence of ATP. The contribution of endogenous PtdInsP2 and PtdInsP to the generation of inositol phosphates was examined in membranes from [3H]inositol-prelabeled brain slices by adding unlabeled Ins1,4,5P3 to trap [3H]Ins1,4,5P3 generated upon breakdown of [3H]PtdInsP2. The maximal rate of [3H]Ins1,4,5P3 appearance was attained in the presence of 150-200 microM added Ins1,4,5P3 and represented 12.5% of the combined rates of formation of [3H]Ins1,4,5P3 and [3H]Ins1,4P2, similar to the content of [3H]PtdInsP2 relative to total 3H-polyphosphoinositides. The results show that, while endogenous PtdIns is not degraded by phospholipase C, the enzyme appears to be equally effective to cleave endogenous PtdInsP2 and PtdInsP.

Animals↗
PubMed1993

Transmembrane signaling through phospholipase C in human cortical membranes.

Stimulation of phosphoinositide-specific phospholipase C (PLC) by carbachol, dopamine and serotonin was measured by supplying exogenous [3H]phosphatidylinositol 4,5-bisphosphate to membranes prepared from human cortex dissected and frozen at autopsy. Subjects with Alzheimer's disease, Parkinson's disease or schizophrenia were compared to age-matched controls with no known neurological disorders. Stimulation of PLC by the neurotransmitters was dependent on the presence of GTP gamma S. Carbachol elicited the greatest stimulations of PLC followed by serotonin and then dopamine. The maximal stimulations of PLC evoked by a neurotransmitter were similar for the various categories of subjects except in Parkinson's patients, where dopamine failed to stimulate PLC beyond the activity attained with carbachol. In the presence of carbachol, the sensitivity of PLC to GTP gamma S was significantly increased in Alzheimer's membranes, but not in age-matched controls or Parkinson's. Overall, the experiments demonstrate the feasibility for using the exogenous substrate assay to study the functionality of the phosphoinositide transmembrane signaling system in human brain.

Cerebral Cortex↗
PubMed1993

Denervation hypersensitivity of histamine H1-receptors in rat brain cortex.

We have studied the effects of the unilateral electrolytic lesion of the medial forebrain bundle at the level of the lateral hypothalamus on the density and functionality of histamine H1 receptors in rat brain cortex. The treatment resulted, after two and four weeks, in an increase in the maximal phosphoinositide breakdown induced by histamine, which can be accounted for by the appearance of a higher potency component for the response. On the other hand, the density of cortical histamine H1 receptors, determined by the specific binding of [3H]mepyramine to membranes, remained unchanged two weeks after the lesion but after four weeks a small but significant increase was also found. These results suggest that the denervation hypersensitivity developed may initially be the result of a more efficient coupling of the H1 receptors to the effector system prior to the subsequent increase in receptor numbers.

Animals↗
PubMed1993

Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices.

Muscarinic cholinergic and alpha 1-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'-O-(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H-inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [3H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the alpha 1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single alpha 1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain alpha 1 adrenoceptors involves an increase in intracellular calcium concentration.

Animals↗
PubMed1992

Quisqualate-stimulated phosphatidylinositol breakdown in rat cerebellar membranes.

The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose-dependent manner in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Half-maximal activation of the quisqualate response required 0.15 microM GTP gamma S and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositides 4,5-bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as L-glutamate, trans-D,L-1-aminocyclopentyl-1,3-dicarboxylic acid, and ibotenate, but not N-methyl-D-aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide-specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.

Amino Acids↗
PubMed1992

Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes.

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.

Animals↗
PubMed1991

Altered phosphoinositide-specific phospholipase C and adenylyl cyclase in brain cortical membranes of cats with GM1 and GM2 gangliosidosis.

Phosphoinositide-specific phospholipase C and adenylyl cyclase were studied in brain cortical membranes from cats with GM1 and GM2 gangliosidosis. In contrast to brain cortical membranes from unaffected control cats, phospholipase C acting against exogenously supplied phosphoinositide substrates did not respond to stimulation by GTP gamma S, carbachol or fluoroaluminate in cortical membranes of cats with gangliosidosis. However, the enzyme was activated by calcium in membranes from affected cats to the same extent as in membranes from control cats. Basal adenylyl cyclase activity was increased 3-fold in cortical membranes of cats with GM1 and GM2 gangliosidosis, compared with unaffected sibling controls. Fluoroaluminate was equally effective in stimulating adenylyl cyclase in controls and in membranes of affected and normal cats. In addition, GppNHp was able to inhibit the forskolin-activated enzyme both in membranes from cats with gangliosidosis and sibling controls. These data suggest that the activation of phosphoinositide-specific phospholipase C in brain membranes by guanine nucleotide binding proteins is markedly impaired in GM1 and GM2 gangliosidoses.

Adenylyl Cyclases↗