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E Claro

Publications and source records attributed to E Claro.

27 records · Page 2Linked to original sources

Dual effect of fluoride on phosphoinositide metabolism in rat brain cortex. Stimulation of phospholipase C and inhibition of polyphosphoinositide synthesis.

We have studied the effects of fluoride, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and carbachol on phospholipase C and polyphosphoinositide synthesis. The experimental system consisted of membranes from rat brain cortex, with exogenous [3H]phosphatidylinositol ([3H]PtdIns) as substrate. In such systems, we have not found evidence to support carbachol and/or GTP[S] stimulation of PtdIns phosphorylation. Fluoride inhibited synthesis of PtdIns4P and PtdIns(4,5)P2 from PtdIns. Consequently, under conditions where breakdown of polyphosphoinositides by phospholipase C was dependent on PtdIns kinase activity, fluoride inhibited activation by GTP[S] plus carbachol of phospholipase C. When conditions allowed direct breakdown of PtdIns and precluded PtdIns kinase activity, the stimulatory effects of fluoride and GTP[S] plus carbachol on phospholipase C activity were additive.

1-Phosphatidylinositol 4-Kinase↗

A novel role for dopamine: inhibition of muscarinic cholinergic-stimulated phosphoinositide hydrolysis in rat brain cortical membranes.

Dopamine inhibited phosphoinositide breakdown as stimulated by carbachol in rat brain cortical membranes. The IC50 value was 14 +/- 2 microM for dopamine's inhibition of phosphatidylinositol hydrolysis as stimulated by 1 mM carbachol. The inhibition was found at low (0.1 microM), but not high (greater than 0.3 microM), concentrations of the non-hydrolyzable guanine nucleotide analog, GTP gamma S. Pharmacological characterization of the response indicated that the dopamine effects were mediated by D1 receptors. The assay conditions precluded any involvement of cyclic-AMP as a mediator of the dopamine response, and thus, a novel role is proposed for dopamine in cortex working through D1 receptors to inhibit phosphoinositide degradation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

Comparison of serotoninergic to muscarinic cholinergic stimulation of phosphoinositide-specific phospholipase C in rat brain cortical membranes.

Stimulation of phosphoinositide-specific phospholipase C (PLC) by muscarinic cholinergic and serotoninergic agonists was measured in rat brain cortical membranes by using exogenously supplied substrates. Serotonin, tryptamine, 5-fluorotryptamine and 5-methyltryptamine stimulated PLC with EC50 values of 1.7, 11.2, 15.0, and 29.4 microM, respectively. Maximal PLC stimulation by serotoninergic agonists, which were all equally efficacious, was about 30% of that attained by carbachol. Ketanserin blocked serotoninergic but not cholinergic activation of PLC, whereas, conversely, atropine blocked the latter but not the former response. The rank order of potency for muscarinic agonists was oxotremorine-M greater than pilocarpine = arecoline greater than carbachol = bethanecol. Unlike the case with tissue slices, all of these muscarinic agonists exhibited full efficacy in this assay of PLC stimulation. Activation of PLC by the neurotransmitters or their analogs was dependent on the addition of guanosine 3'-O-thiotriphosphate (GTP gamma S). Stimulation of PLC by GTP gamma S alone or in combination with 5-methyltryptamine had an apparent EC50 of about 0.4 microM. However, when carbachol or other muscarinic agonists were used, the EC50 for GTP gamma S was significantly lower. We have previously shown that dopamine working through D1 receptors inhibits the PLC response to carbachol by preventing this shift in the apparent EC50 for GTP gamma S. Dopamine did not have a similar effect on 5-methyltryptamine stimulation of PLC. The results indicate that the postreceptor mechanisms of PLC activation are distinct for muscarinic as opposed to serotoninergic agonists in brain cortex.

Animals↗

Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes.

The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTP gamma S (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 microM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 microM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTP gamma S was greatest on a percentage basis at the lowest free Ca2+ concentrations. Above 1 microM free Ca2+, the stimulatory effect was lost, whereas 10 microM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 microM. The best stimulation of PI breakdown due to carbachol plus GTP gamma S was seen with 0.3 microM free Ca2+ and 100 microM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 microM GTP gamma S alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 microM GTP gamma S. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTP gamma S in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

Adenosine Triphosphate↗

Differential effects of chronic treatment with mianserin and protryptiline on rat brain cortical alpha 1-adrenoceptors.

Chronic administration of mianserin induced an increase in the density of [3H] prazosin binding sites (23%) in membranes and in the maximal noradrenaline stimulation of phosphoinositide breakdown (81%) in slices from rat brain cortex. In contrast, a similar treatment with protryptiline did not induce any significant changes. These findings suggest that the effects of antidepressant drugs on rat brain cortical alpha 1-adrenoceptors may depend on the characteristics of the drug used.

Animals↗

Carbachol and histamine stimulation of guanine-nucleotide-dependent phosphoinositide hydrolysis in rat brain cortical membranes.

Guanine nucleotides have been shown to stimulate phosphoinositide breakdown in brain membranes, but no potentiation of such an effect by agonist was demonstrated. We have studied the effect of carbachol and histamine on guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulation of inositol phosphates formation in [3H]inositol-labelled rat brain cortical membranes. In this preparation, GTP[S] enhancement of phosphoinositide hydrolysis required the presence of MgATP and low Ca2+ concentration (100 nM). Carbachol potentiation of the GTP[S] effect was only observed when 1 mM-deoxycholate was also added. Under these conditions, stimulated production of [3H]inositol phosphates was linear for at least 15 min, and [3H]inositol bisphosphate [( 3H]IP2) accounted for approx. 80%, whereas the amount of [3H]inositol trisphosphate [( 3H]IP3) was very low. Stimulation by GTP[S] was concentration-dependent (half-maximal effect at 0.86 microM), and its maximal effect (815% over basal) was increased by 1 mM-carbachol (1.9-fold) and -histamine (1.7-fold). Both agonists decreased the slope index of the GTP[S] concentration/effect curve to values lower than unity, suggesting the appearance of some heterogeneity in the population of guanine-nucleotide-binding proteins (G-proteins) involved. The carbachol and histamine effects were also concentration-dependent, and were inhibited by atropine and mepyramine respectively. Fluoroaluminate stimulated phosphoinositide hydrolysis to a higher extent than GTP[S] plus carbachol, and these stimulations were not additive, indicating that the same polyphosphoinositide phospholipase C-coupled G-protein mediates both effects.

Aluminum↗

Histamine-stimulated phosphoinositide hydrolysis in developing rat brain.

Histamine stimulation of phosphoinositide breakdown has been studied in brain cortex from developing rats by measuring the accumulation of [3H]inositol monophosphate in lithium-treated slices prelabeled with [3H]inositol. Histamine stimulated phosphoinositide hydrolysis as early as day 3 after birth. The maximal response increased about 3-fold up to day 15 and remained essentially at the same level until adulthood. This developmental pattern contrasted with the 8-fold increase in [3H]mepyramine binding to histamine H1 receptors which takes place from the first to the end of the fourth week of life. Nonlinear regression analysis of concentration-effect curves for histamine generated at 6, 10, and 15 days of age revealed the presence of two components in the response, whereas a single component was found in adults. A high potency component showed EC50 values increasing from 6.4 microM at day 6 to 26.5 microM at day 15, and its contribution to the maximal response augmented from 65.8% to 78.9% during the same period. In 6-day-old animals, the H1 receptor agonists N alpha-methylhistamine and N alpha,N alpha-dimethylhistamine produced single-component responses with maximal effects similar to that estimated for the high potency component for histamine, and their relative potencies were consistent with an H1 receptor-mediated response. Inhibition constants for mepyramine, calculated from the parallel displacement of the concentration-response curves for N alpha-methylhistamine and the high potency component for histamine (6.3 nM and 10.8 nM, respectively) closely agreed with the reported affinity of rat brain H1 receptors for this compound. The low potency component of the response (EC50 = 175 microM at day 6 and EC50 = 260 microM at day 15) did not involve histamine-H2, serotonin 5-HT2, alpha 1-adrenergic, or muscarinic receptors. Pretreatment of 6-day-old cortical slices with the irreversible antagonist phenoxybenzamine, at concentrations that completely abolish [3H]mepyramine binding, left about 25% of the response to 1 mM histamine unaffected. This portion of the response, which was, moreover, insensitive to 1 microM mepyramine, was of a magnitude similar to that of the low potency component. Thus, at early stages of rat brain development, histamine induces phosphoinositide hydrolysis in part through a mechanism different from activation of H1 receptors. The loss of histamine potency and efficacy at H1 receptors throughout development is discussed and the natural occurrence of H1 receptor subsensitivity suggested.

Aging↗

Phosphoinositide hydrolysis mediated by histamine H1-receptors in rat brain cortex.

Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of lithium in [3H]inositol-prelabelled slices from rat brain cortex in a concentration-dependent manner, with an EC50 value of 94.7 microM. High concentrations of antagonists of histamine H2 receptors, muscarinic receptors, alpha 1-adrenoceptors and serotonin receptors did not inhibit the effect. The histamine H1-receptor antagonists mepyramine, triprolidine, promethazine, d-chlorpheniramine and the tricyclic antidepressant doxepin inhibited the response with Ki values corresponding to an interaction with histamine H1-receptors. The EC50 for the response was about three times lower than the Ki value (approximately 300 microM) for the inhibition by histamine of [3H]mepyramine binding to membranes from rat brain cortex. Partial inactivation of H1-receptors with the alkylating antagonist phenoxybenzamine resulted in similar reductions in [3H]mepyramine binding sites and in the maximal histamine-induced [3H]inositol 1-phosphate accumulation, without affecting the KD for the radioligand or the EC50 for the response. The apparent dissociation constant for histamine calculated from these experiments (KA = 92.2 microM) was not different from the EC50 value. The present results indicate that histamine-stimulated phosphoinositide hydrolysis in rat brain cortex is mediated by H1-receptors and that no receptor reserve is present.

Animals↗

[3H]mepyramine binding to histamine H1 receptors in bovine retina.

The promethazine-sensitive [3H]mepyramine binding was used to determine the presence of histamine H1 receptors in membranes from bovine retina. Specific mepyramine binding to retinal membranes was reversible, saturable and of high affinity. The apparent dissociation constant (KD = 2.2 +/- 0.4 nM) and the density of binding sites (Bmax = 60.9 +/- 5.1 fmol/mg protein), obtained in equilibrium studies, were similar to those found in bovine brain cortex. Binding was stereospecific and the inhibitory potencies of H1 and H2 antagonists indicated that [3H] mepyramine binding sites in the retina have characteristics of H1 receptors.

Aminopyridines↗