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PubMed2002

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex.

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Adaptor Protein Complex delta Subunits↗

PubMed2003

Kinetic analysis of receptor-activated phosphoinositide turnover.

We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2-3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCdelta1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

Bradykinin↗

PubMed1990

Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

Angiotensins↗

PubMed2006

Phosphoinositide 3-Kinase C2beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms.

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans.

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.

Amino Acid Sequence↗

PubMed1990

Self suppression of phosphoinositide turnover and contraction by stimulating release of endogenous endothelium derived relaxing factor in vascular action of histamine.

STUDY OBJECTIVE: The aim of the study was to examine the relationship between phosphoinositide turnover and vascular contraction in response to histamine, and the role of endothelium derived relaxing factor (EDRF) in these activities. DESIGN: Thoracic aortic strips were studied under isometric tension in an organ bath. Phosphoinositide turnover was studied using [32P]Pi labelling. Cumulative concentration-response curves were obtained for histamine, and for histamine plus H1 and H2 antagonists. The effect of endothelial denudation was examined, as was that of removing Ca2+ from buffer medium, and of adding methylene blue, an inhibitor of EDRF. EXPERIMENTAL PREPARATIONS: Thoracic aortic rings were obtained from 81 male Japanese white rabbits, weight 2.0-2.4 kg. MEASUREMENTS AND MAIN RESULTS: Histamine (0.1 mumol-0.1 mmol.litre-1) caused concentration dependent contractions and increases of [32P]Pi incorporation into phosphatidylinositol (ED50 = 4.6 mumol.litre-1 and 4.0 mumol.litre-1 respectively). Diphenhydramine inhibited contractile response and phosphatidylinositol labelling by histamine. Cimetidine potentiated contractile response but had no effect on phosphatidylinositol labelling. Endothelial denudation potentiated maximum contraction by 15% and augmented phosphatidylinositol labelling at 0.1 mmol.litre-1 histamine by 30%. In intact aortic rings, methylene blue (10 mumol.litre-1), an inhibitor of EDRF, potentiated histamine induced phosphatidylinositol labelling, but had no influence on denuded rings. A-23187 (0.01 mumol.litre-1), which caused release of EDRF from intact endothelium, suppressed histamine induced phosphatidylinositol labelling in intact aortic rings. CONCLUSIONS: The results suggest that stimulation of the release of EDRF in response to histamine causes self suppression of phosphoinositide turnover and contraction.

Animals↗

PubMed1993

Diet restriction prevents aging-induced deficits in brain phosphoinositide metabolism.

Receptor-activated hydrolysis of [3H]inositol-labeled phosphoinositides was evaluated in brain slices of 6-month-old (6M) and 24-month-old (24M) F344 rats that had been fed ad libitum (AL) or restricted (60% of normal) diet (RD). The muscarinic cholinergic agonist, carbachol, and the dopamine receptor agonist, SKF3893, stimulated significantly lower accumulation of [3H]inositol phosphates in striatal and cortical slices of the 24M AL rats compared to the 6M AL group. This observation suggests a decreased capacity of the aged brain to respond to receptor-activated phosphoinositide hydrolysis. Furthermore, the 24M RD tissues gave significantly higher responses to carbachol (p < .01) or SKF38393 (p < .01) compared to the 24M AL group. The 24M RD responses were not significantly different from corresponding responses in the 6M AL rats, implying complete prevention of the aging effect in the diet-restricted animals up to at least 24 months of age. Concomitant observations of decreased phosphoinositide labeling in the 24M AL tissues and prevention of this decrease in diet restriction may contribute to the observed effects of aging on inositol phosphate formation.

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

PubMed1994

Lithium potentiates phosphoinositide-linked 5-HT receptor stimulation in vivo.

The therapeutic effect of lithium in manic-depressive illness may involve alterations in the activity of the phosphoinositide second messenger system. Lithium administration to rats potentiates responses to cholinergic agonists, as evidenced by the production of seizures in lithium-treated rats after normally nonconvulsant doses of cholinergic agonists. We now report that lithium also potentiates the response to a serotonin (5-HT) agonist, DOI, that activates 5-HT2/5-HT1C receptors coupled to phosphoinositide hydrolysis. EEG recordings showed that administration of DOI (8 mg kg-1) to lithium-treated, but not to lithium-naive, rats caused seizures which were blocked by ritanserin pretreatment. These results demonstrate that lithium pretreatment causes a normally subconvulsive dose of serotonergic, in addition to cholinergic, agonists to induce seizures. Since DOI, like cholinergic agonists, activates receptors coupled with phosphoinositide hydrolysis and lithium potentiates responses to each, this second messenger system is likely to be involved in this effect of lithium.

Amphetamines↗

PubMed1989

Endothelin-1 activates the phosphoinositide cascade in cultured glomerular mesangial cells.

The newly described peptide, endothelin-1 (ET-1), causes profound vasoconstriction, but the pathways of transmembrane signaling remain unclear. We demonstrate that in glomerular mesangial cells, smooth muscle-like vascular pericytes, ET-1 elevates intracellular Ca2+ ([ Ca2+]i) by activating the phosphoinositide cascade. ET-1 increased [Ca2+]i in two distinct kinetic patterns. Concentrations of 0.1-10.0 pM ET-1 caused a slow but sustained increase in [Ca2+]i that was insensitive to voltage-gated Ca2+ channel blockade but dependent on extracellular Ca2+. In contrast, ET-1 greater than or equal to 0.1 nM evoked a rapid, transient increase in [Ca2+]i followed by a lesser, sustained increase. Only the sustained increment of [Ca2+]i required extracellular Ca2+, but both phases were unaffected by Ca2+ channel blockade. The transient increase in [Ca2+]i resulted from activation of phosphoinositide-specific phospholipase C to release inositol trisphosphate (IP3), which mobilizes Ca2+ from intracellular stores. ET-1 also stimulated amiloride-inhibitable Na+/H+ exchange, causing cytosolic alkalinization. Thus, the phosphoinositide cascade probably mediates some biological functions of ET-1, including possibly contraction via pharmacomechanical coupling.

Animals↗

PubMed1996

Structural and functional diversity of phosphoinositide 3-kinases.

Phosphoinositide 3-kinases (PI3-kinases) have been shown to be recruited to cell surface receptor signal complexes whose formation is triggered by growth factors, cytokines and other ligands. PI3-kinases are also involved in protein sorting phenomena. A number of PI3-kinase isotypes have been characterised in several laboratories. Here the relations between the PI3-kinases, PI4-kinases and PI5-kinases and other potential phosphoinositide kinases are analysed. A study of the relation of structure to function for sequence motifs defined through the use of homology searches and protein modelling techniques is described and used to assign the family of phosphoinositide kinases to subgroups.

Amino Acid Sequence↗

PubMed1990

Time-related Changes in Basal Phosphoinositide Turnover after Induction of Long-term Potentiation in the Dentate Gyrus are Blocked by Commissural Stimulation.

We have examined basal phosphoinositide turnover in synaptosomes obtained from the dentate gyrus of anaesthetized rats in which long-term potentiation was induced unilaterally in perforant path-granule cell synapses. Relative to the unpotentiated side, [3H]myo-inositol labelling of inositol phosphates was significantly enhanced 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. Similarly, [14C]arachidonic acid labelling of 1,2-diacylglycerol was increased 45 min and 3 h after induction of long-term potentiation, but reduced after 2.5 min. In a second series of experiments, induction of long-term potentiation was blocked by stimulation of the commissural projection to granule cells. In synaptosomes prepared from this tissue, there was no difference in phosphoinositide turnover between tetanized and control sides at any of the three post-tetanic intervals. We conclude that in the dentate gyrus, long-term potentiation is associated with an increase in phosphoinositide turnover which is established between 2.5 min and 45 min post-tetanus and which persists for at least 3 h.

Journal Article↗

PubMed1985

Effects of systemically administered lithium on phosphoinositide metabolism in rat brain, kidney, and testis.

A single subcutaneous dose of 10 mEq/kg LiCl gives rise to an increase in the cerebral cortex level of myo-inositol-1-P (I1P) that closely follows cortical lithium levels and, at maximum, is 40-fold above the control value. Kidney and testis show smaller increases in I1P level following LiCl administration. The I1P level is still sixfold greater than that of untreated rat cortex 72 h later. In cortex, parallel increases also occur in myo-inositol-4-P (I4P) and myo-inositol 1,2-cyclic-P (cI1,2P), whereas myo-inositol-5-P (I5P) remains unchanged. The cortical increases in I1P and I4P levels are partially reversed by administering 150 mg/kg of atropine 22 h after the LiCl, treatment that does not affect cI1,2P. When doses of LiCl from 2 to 17 mEq/kg are given, the cerebral cortex levels of I1P and myo-inositol, measured 24 h later, are found to reach a plateau at about 9 mEq/kg of LiCl, whereas cortical lithium levels continued to increase with greater LiCl doses. Levels of all three of the brain phosphoinositides are unchanged by the 10 mEq/kg LiCl dose, as is the uptake of 32Pi into these lipids. Chronic dietary administration of LiCl for 22 days showed that the effects of lithium on I1P and myo-inositol levels persist for that period. Over the course of the chronic administration of the lithium, levels of I1P, myo-inositol, and of lithium in cortex remained significantly correlated. We believe that these increases in inositol phosphates result from endogenous phosphoinositide metabolism in cerebral cortex and that lithium is capable of modulating that metabolism by reducing cellular myo-inositol levels. The size of the effect is a function of both lithium dose and the degree of stimulation of receptor-linked phosphoinositide metabolism. This property of lithium may explain part of its ability to moderate the symptoms of mania. Our chronic study suggests that prolonged administration of LiCl does not result in compensatory changes in myo-inositol-1-P synthase or myo-inositol-1-phosphatase.

Administration, Oral↗

PubMed1987

Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells.

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.

Animals↗

PubMed1987

Enhanced coupling of neonatal muscarinic receptors in rat brain to phosphoinositide turnover.

The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo-[2-3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day-18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1-10 mM) was at the adult level (i.e., three-fold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose-response curve was shifted to the left, giving a half-maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine-2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of "spare" muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.

Aging↗

PubMed1987

Stimulation of phosphoinositide hydrolysis by serotonin in C6 glioma cells.

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Modulation of phosphoinositide hydrolysis by NaF and aluminum in rat cortical slices.

NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 mM NaF. Addition of 10 or 100 microM AlCl3 or aluminum maltol did not alter the effect of NaF, whereas at 500 microM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 mM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 microM phorbol 12-myristate 13-acetate, a phorbol ester, inhibited carbachol-induced, but not NaF-induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.

Aluminum↗

PubMed1990

Interaction of sigma-compounds with receptor-stimulated phosphoinositide metabolism in the rat brain.

Sigma-receptors are nonopioid, nondopaminergic receptors that bind with high affinity several antipsychotic drugs and appear to be involved in regulation of posture and movement. The second messenger system coupled to these receptors is still unknown. Recently, an inhibitory effect of various sigma-compounds on carbachol-stimulated phosphoinositide metabolism has been reported. We have investigated the effect of six sigma-compounds on carbachol- and norepinephrine-stimulated 3H-inositol phosphate accumulation in rat cerebral cortex slices. All compounds tested had a dose-dependent inhibitory effect on both systems, although their order of potency differed between neurotransmitters. Pentazocine and 1,3-di-o-tolylguanidine were the most potent inhibitors of carbachol-stimulated phosphoinositide turnover (IC50 = 31.5 and 45.7 microM, respectively), while haloperidol showed the greatest potency on the norepinephrine-coupled system (IC50 = 3.5 microM). In the presence of IC50 concentrations of these inhibitors, the dose-response curves for the agonists were shifted to the right and the EC50 values were significantly increased. Sigma-compounds also inhibited the binding of [3H]quinuclidinyl benzilate to muscarinic receptors and of [3H]prazosin to alpha 1-adrenoceptors in cortical membranes. In the presence of IC50 concentration (11 microM) of 1,3-di-o-tolylguanidine, no differences were found in the maximal number of muscarinic binding sites, whereas the dissociation constant increased approximately fivefold, indicating a decrease of the radioligand's affinity for the receptor. These results indicate that sigma-compounds, at micromolar concentrations, inhibit muscarinic and alpha 1-adrenergic receptor-coupled phosphoinositide metabolism, probably through an interaction with the neurotransmitter recognition sites.

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↗