Stimulation Of Phosphoinositide Hydrolysis Research Articles

Effects of ibogaine and noribogaine on phosphoinositide hydrolysis

The effects of the antiaddictive compound, ibogaine, and its primary metabolite, noribogaine (12-hydroxyibogamine), on phosphoinositide hydrolysis were investigated. Although ibogaine did not alter phosphoinositide turnover in either striatal or hippocampal slices, noribogaine elicited a concentration-dependent increase in the generation of [3H]inositol phosphates. This stimulation was not altered by inclusion of tetrodotoxin, cadmium or ω-conotoxin indicating that the increased production of [3H]inositol phosphates was not secondary to a release of one or more neurotransmitters. The present study indicates a stimulation of phosphoinositide hydrolysis by noribogaine may be involved in the behavioral effects of ibogaine.

Effects of ibogaine and noribogaine on phosphoinositide hydrolysis

The effects of the antiaddictive compound, ibogaine, and its primary metabolite, noribogaine (12-hydroxyibogamine), on phosphoinositide hydrolysis were investigated. Although ibogaine did not alter phosphoinositide turnover in either striatal or hippocampal slices, noribogaine elicited a concentration-dependent increase in the generation of [ 3H]inositol phosphates. This stimulation was not altered by inclusion of tetrodotoxin, cadmium or ω-conotoxin indicating that the increased production of [ 3H]inositol phosphates was not secondary to a release of one or more neurotransmitters. The present study indicates a stimulation of phosphoinositide hydrolysis by noribogaine may be involved in the behavioral effects of ibogaine.

Pharmacological characterization of desensitization in a human mGlu1 alpha-expressing non-neuronal cell line co-transfected with a glutamate transporter.

1. Stimulation of phosphoinositide hydrolysis by human mGlu1 alpha (HmGlu1 alpha) was examined in a non-neuronal cell line (AV12-664) co-expressing both HmGlu1 alpha and a rat glutamate/aspartate transporter (GLAST). 2. Desensitization of HmGlu1 alpha could be elicited by inhibition of the GLAST transporter with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). Maximal inhibition of HmGlu1 alpha-mediated phosphoinositide hydrolysis was induced upon 24 h pretreatment with trans-PDC. The concentration of glutamate in the extracellular medium also rose significantly in cells pretreated with trans-PDC. Glutamate levels increased upon incubation with trans-PDC in a time-dependent manner, with maximal glutamate levels attained after 24 h incubation with trans-PDC. 3. The time required for desensitization of HmGlu1 alpha by trans-PDC was compared to the time course for desensitization elicited by the direct-acting mGlu receptor agonists, 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) and (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG). Both direct-acting mGlu receptor agonists elicited desensitization of HmGlu1 alpha more rapidly than did trans-PDC, with maximal inhibition of agonist-induced phosphoinositide hydrolysis upon 12 h pretreatment. Agonist-induced desensitization could be fully reversed upon washout of agonist for 12 h. 4. Both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha could be blocked by inclusion of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an mGlu receptor antagonist, in the pretreatment medium. 5. Agonist-stimulated phosphoinositide hydrolysis by HmGlu1 alpha was found to parallel closely agonist-induced desensitization of HmGlu1 alpha. Thus, the EC50 values for 1S,3R-ACPD- and 3,5-DHPG-stimulated phosphoinositide hydrolysis were similar to the EC50 values for eliciting desensitization of HmGlu1 alpha. 6. These studies demonstrate desensitization of recombinant human mGlu1 alpha receptor in a non-neuronal cell line in which the receptor can be regulated by direct activation or by manipulation of glutamate transporter activity. Desensitization of HmGlu1 alpha was found to be mediated by activation of the receptor since the mGlu receptor antagonist, MCPG, blocked both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha. Furthermore, agonist-induced desensitization of HmGlu1 alpha was found to parallel receptor-mediated stimulation of phosphoinositide hydrolysis.

Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms.

Angiotensin II (ANG II) elicits an ANG II type 1 (AT1) receptor-mediated decrease in voltage-dependent K+ current (Ik) and an increase in voltage-dependent Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem. Modulation of these currents by ANG II involves intracellular messengers that result from an AT1 receptor-mediated stimulation of phosphoinositide hydrolysis. For example, the effects of ANG II on IK and ICa were abolished by phospholipase C antagonists. The reduction in IK produced by ANG II was attenuated by either protein kinase C (PKC) antagonists or by chelation of intracellular Ca2+. By contrast, PKC antagonism abolished the stimulatory effect of ANG II on ICa. Superfusion of the PKC activator phorbol 12-myristate 13-acetate produced effects on IK and ICa similar to those observed after ANG II. Furthermore, intracellular application of inositol 1,4,5-trisphosphate (IP3) elicited a significant reduction in IK. This suggests that the AT1 receptor-mediated changes in neuronal K+ and Ca2+ currents involve PKC (both IK and ICa) and IP3 and/or intracellular Ca2+ (IK).

Regulation of Na +,K +-ATPase activity by dopamine in cultured rat aortic smooth muscle cells

We investigated the effect of dopamine on Na +,K +-ATPase activity in cultured aortic smooth muscle cells. Na +,K +-ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D 1 receptor agonist) and quinpirole (a D 2 receptor agonist) produced 62%, 50% and 49% inhibition of Na +,K +-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na +,K +-ATPase inhibition was blocked by selective receptor antagonists. The Na +,K +-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na +,K +-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-γ-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D 1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na +,K +-ATPase activity through specific vascular receptors. Dopamine D 1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na +,K +-ATPase activity in response to dopamine D 1 receptor activation may be mediated by the phospholipase C signaling pathway.

Stimulation of Phosphoinositide Hydrolysis by γ- and δ-Hexachlorocyclohexane in Primary Cultures of Cerebellar Granule Cells: Interaction with Glutamate and Carbachol Receptor-Mediated Phosphoinositide Response and Effects of Specific Pharmacological Agents

The accumulation of [3H]inositol phosphates derived from phosphoinositide hydrolysis stimulated by γ- and δ-hexachlorocyclohexane isomers was characterized in primary cultures of cerebellar granule cells. The EC50for γ- and δ-hexachlorocyclohexane was 106 and 85 μM,respectively. Stimulatory effects of hexachlorocyclohexane isomers were highly dependent on extracellular Ca2+but they were not inhibited by classical voltage-sensitive Ca2+and Na+channel blockers. The Na+/Ca2+exchanger blocker amiloride caused a significant inhibition of δ-hexachlorocyclohexane effects. A lack of additive effects on phosphoinositide hydrolysis stimulation between hexachlorocyclohexane isomers and depolarization by high K+was observed. The effects of each hexachlorocyclohexane isomer on glutamate or carbachol-induced inositol phosphate stimulation were also not additive, whereas that of high K+was less than additive or synergistic when combined with glutamate or carbachol, respectively. When neuronal cells were exposed to the combination of δ-hexachlorocyclohexane and glutamate or carbachol in the presence of the respective receptor antagonists only the δ-hexachlorocyclohexane stimulatory effect was observed. Thus, the inhibition of glutamate- and carbachol-stimulated phosphoinositide hydrolysis by δ-hexachlorocyclohexane seems to imply a receptor-independent mechanism. It is suggested that both γ- and δ-hexachlorocyclohexane activate phosphoinositide-specific phospholipase C partly through Ca2+-related mechanisms.

Metabotropic glutamate receptor subtype 1a activates adenylate cyclase when expressed in baby hamster kidney cells

1. 1. The coupling of subtype mGluR1a of the metabotropic glutamate receptor family to phosphoinositide hydrolysis, cAMP-formaaon and arachidonic acid release was characterized when the receptor was expressed in baby hamster kidney (BHK) cells. 2. 2. When measuring cAMP-formation in BHK cells expressing mGluR1a, the rank order of agonist potency as well as the efficacy of mGluR1a antagonists was comparable to what has been observed when measuring mGluR 1a-mediated phosphoinositide (PI)-hydrolysis. 3. 3. However, while the presence of extracellular calcium increased the efficacy of quisqualate to stimulate phosphoinositide hydrolysis no significant effects were observed when measuring quisqualate-induced cAMP-formation. 4. 4. Pretreatment of mGluR 1a-expressing cells with pertussis toxin increased quisqualate-induced cAMP-formation in contrast to the observed partial inhibition of PI-hydrolysis by pertussis toxin. Cholera toxin increased cAMP-formadon in BHK cells but showed no effects on PI-hydrolysis. 5. 5. While quisqualate also stimulated [ 3H]-arachidonic acid release from BHK cells expressing mGluR1a this effect may be secondary to activation of phospholipase C. 6. 6. These data further suggest that mGluR1a is coupled to PI-hydrolysis as well as cAMP-formation via different G-proteins which can be discriminated by their sensitivity to pertussis toxin.

Endothelin-Dependent Actions in Cultured AT-1 Cardiac Myocytes

The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC)-epsilon immunoreactivity from the soluble to the particulate cell fraction. The subcellular distributions of PKCalpha and PKCzeta (also expressed by AT-1 cells) are not influenced by endothelin. Using quantitative fluorescence microscopy with fura 2, we examined the effects of endothelin on intracellular calcium. In electrically driven myocytes, endothelin induces a rapid and transient increase in the amplitude of the calcium transient. This is blocked by both phorbol 12-myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKCepsilon plays a critical role in endothelin receptor-dependent increases in intracellular calcium. Endothelin also stimulates mitogen-activated protein kinase (MAPK). MAPK activation is markedly attenuated by pretreatment with PMA or pertussis toxin (PTX, to activate susceptible G protein alpha subunits); it is completely prevented by combined pretreatment with PMA and PTX. In contrast, it is not attenuated by chelation of intracellular calcium with BAPTA. These findings indicate that the pathway for endothelin receptor stimulation of MAPK involves PKCepsilon and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKCepsilon as a mediator of endothelin receptor-dependent increases in cytosolic calcium and MAPK activity in AT-1 cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKCepsilon and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes.

Thrombin receptor actions in neonatal rat ventricular myocytes.

Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor-derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels.

Activities of novel aryloxyalkylimidazolines on rat 5-HT 2A and 5-HT 2C receptors

Using transfected NIH 3T3 mouse fibroblast cell lines expressing the rat 5-HT 2A and rat 5-HT 2C receptor subtypes, and techniques of 2-[ 125I](+)-iodolysergic acid diethylamide ([ 125I]LSD) binding and serotonin (5-hydroxytryptamine, 5-HT)-stimulated phosphoinositide hydrolysis, we have characterized a new structural class of 5-HT receptor ligands, the aryloxyalkylimidazolines. These compounds were found to be potent competitors of [ 125I]LSD binding at both receptor subtypes ( K i ∼ 5–200 nM) and to have efficacy ranging from potent competitive antagonists (IC 50 ∼ 25 nM) to moderately potent full agonists (EC 50 ∼ 200 nM). Some of these compounds are agonists at both receptor subtypes, while others are 5-HT 2C receptor agonists with 5-HT 2A receptor antagonist activity. None of the aryloxyalkylimidazolines reported here have 5-HT 2A or 5-HT 2C receptor selective antagonist activity. Since these compounds are novel structures, we compared them with a variety of reference 5-HT receptor ligands selected from other chemical classes that have previously been studied at 5-HT 2A and 5-HT 2C receptors in native tissues.

Dual bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway.

Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis.

Cyclic GMP inhibition of metabotropic glutamate receptor-induced phosphoinositide hydrolysis in mesencephalic neurons

The effect of cGMP on metabotropic glutamate receptor-induced stimulation of phosphoinositide hydrolysis in mesencephalic neuronal cultures was evaluated by cell incubation with the stable analogue dibutyryl-cGMP (10μM). A complete blockade of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- or quisqualate-induced inositol phosphate formation was observed. Ionotropic glutamate receptors in mesencephalic neurons activate cGMP formation and, through this intracellular messenger, they might control mGluR activity.

Inhibition of muscarinic stimulated phosphoinositide hydrolysis in the rat parotid gland by cAMP

The ability of agents that increase or mimic cAMP to affect muscarinic receptor mediated phosphoinositide hydrolysis was investigated in the rat parotid gland. Forskolin (10 μM) and isoproterenol (10 μM) elevated cAMP in the parotid gland by 2-fold and 7-fold, respectively, and these agents also inhibited oxotremorine-M (3 H-M) mediated phosphoinositide hydrolysis by 14% and 26%, respectively. Forskolin (1,4.3, 18, and 75 μM) increased cAMP accumulation and inhibited PIP 2 hydrolysis in a concentration-dependent manner. Forskolin (75 μM) shifted the concentration-response curve for the full agonist oxotremorine-M rightward by 4.2-fold. Pre-treatment with the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) reduced the maximum effect of oxotremorine-M by 31%. The inhibitory effect of isoproterenol and forskolin on muscarinic receptor-mediated phosphoinositide hydrolysis was unaffected by the removal of extracellular Ca 2+. Moreover, isoproterenol and forskolin dampened sodium fluoride and oxotremorine-M mediated phosphoinositide hydrolysis to the same extent suggesting that the inhibitory effect of cAMP is downstream from the muscarinic receptor.

Cross-desensitization of Chemoattractant Receptors Occurs at Multiple Levels: EVIDENCE FOR A ROLE FOR INHIBITION OF PHOSPHOLIPASE C ACTIVITY

To define the molecular mechanisms of cross-regulation among chemoattractant receptors, we stably coexpressed, in a rat basophilic leukemia (RBL-2H3) cell line, epitope-tagged receptors for the chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide of the fifth component of the complement system (C5a), and interleukin-8 (IL-8). All the expressed receptors underwent homologous phosphorylation and desensitization upon agonist stimulation. When co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of the other. Activation of epitope-tagged fMLP receptor (ET-FR) also cross-phosphorylated ET-C5aR and ET-IL-8RA, but ET-FR was totally resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation of [35S]guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) binding and Ca2+ mobilization were cross-desensitized by each other and by fMLP. Stimulation of [35S]GTP gamma S binding by fMLP was also not cross-desensitized by C5a or IL-8, however, Ca2+ mobilization was, suggesting a site of inhibition distal to G protein activation. Consistent with this desensitization of Ca2+ mobilization, inositol 1,4,5-trisphosphate release in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP and C5a cross-desensitized each other's ability to stimulate phosphoinositide hydrolysis. Taken together, these results indicate that receptor cross-phosphorylation correlates directly with desensitization at the level of G protein activation. The ET-FR was resistant to this process. Of note, cross-desensitization of ET-FR at the level of phosphoinositide hydrolysis and Ca2+ mobilization was demonstrated in the absence of receptor phosphorylation. This suggests a new form of chemoattractant cross-regulation at a site distal to receptor/G protein coupling, involving the activity of phospholipase C.

Effect of age on noradrenaline responses in rat tail artery and aorta: role of endothelium.

1. We have analysed the impact of ageing on the contractile responses induced by noradrenaline on endothelium intact and denuded aorta and tail artery rings from Sprague-Dawley rats. In addition, the influence of age on noradrenaline stimulation of phosphoinositide hydrolysis was investigated. 2. The sensitivity and the phosphatidylinositol hydrolysis to noradrenaline in aorta and tail artery were not modified by age. Intact tail artery rings showed a greater maximal contraction (Emax) to noradrenaline in old as compared to young animals. However, no Emax modification by age was observed in aorta (intact or denuded) and in denuded tail artery rings. 3. Removal of endothelial cells resulted in an increase of noradrenaline sensitivity but not the Emax in aorta from each age group. 4. In contrast, the absence of endothelium did not modify (young rats) or diminish (aged rats) the alpha 1-adrenoceptor-mediated responses in tail artery. 5. These results seem to indicate that: (1) there is no influence of age on noradrenaline responses in presence of endothelium; and (2) responses in denuded preparations seem to indicate a differential role of endothelium on noradrenaline responses obtained in different vascular beds.

Synergistic interaction of glucose and neurohumoral agonists to stimulate islet phosphoinositide hydrolysis

The interaction between neurohumoral agonists and glucose to stimulate phosphoinositide (PI)-specific phospholipase C (PLC) and insulin release was examined. In freshly isolated rat islets, maximal glucose (40 mM), cholecystokinin (CCK; 300 nM), or carbachol (CCh; 1 mM) stimulated PI hydrolysis 6.5-, 9.8-, and 5.7-fold, respectively, above basal. The combination of glucose and CCK or of glucose and CCh, but not of CCK and CCh, synergistically increased PI hydrolysis 23.2- and 21.6-fold, respectively, indicating that these secretagogues activate PLC by distinct pathways and that there is an interaction between them. This synergy was maximal at physiological concentrations of stimulatory glucose (8-10 mM) and was paralleled by a marked synergistic stimulation of insulin secretion. The enhanced PI response was partially Ca2+ dependent and may involve the activation of distinct isozymes of PLC, which we identify in islets. These studies demonstrate for the first time a unique and highly sensitive synergistic interaction between glucose and neurohumoral agonists to stimulate PI hydrolysis, and they suggest that enhanced PI hydrolysis is important in the potentiation of glucose- and neurohumoral-stimulated insulin secretion.

Growth factor upregulation of a phosphoinositide-coupled metabotropic glutamate receptor in cortical astrocytes

CNS function depends on a capacity for plasticity during development, following injury, and in response to changing environmental conditions. Functional alterations in signal transduction pathways and in neurotransmitter receptor expression are possible mechanisms for the expression of such plasticity. In the present report, we demonstrate that exposure of astrocytes to specific growth factors alters both the functional activity and the protein levels of a specific glutamate receptor. Exposure of astrocytes to basic fibroblast growth factor, epidermal growth factor, or transforming growth factor-alpha produced marked increases in the ability of metabotropic glutamate receptor (mGluR) agonists to stimulate phosphoinositide hydrolysis. Using Western immunoblotting, we demonstrate that an increase in the levels of one of the phosphoinositide-coupled mGluR subtypes, mGluR5, accompanies the increased ability of mGluR agonists to stimulate phosphoinositide hydrolysis. In contrast, another phosphoinositide-coupled subtype of this receptor family, mGluR1 alpha, was not present at detectable levels in these cultures. The enhanced stimulation of phosphoinositide hydrolysis showed little sensitivity to pertussis toxin, and appeared to be selective to mGluR agonists, as there was not a similar increase in the ability of norepinephrine or carbachol to stimulate phosphoinositide hydrolysis. These findings demonstrate that expression of mGluRs in astrocytes is plastic, and indicate a novel pathway through which specific growth factors may selectively modulate neurotransmitter action.

Adenosine receptor activation potentiates phosphoinositide hydrolysis and arachidonic acid release in DDT 1-MF2 cells: Putative interrelations

Studies were undertaken in an effort to discern possible mechanisms by which the A 1 adenosine receptor agonist cyclopentyladenosine (CPA) enhances the norepinephrine-stimulated (NE-stimulated) hydrolysis of phosphoinositides in DDT 1-MF2 cells. Measurements of arachidonic acid release revealed similar behaviours to those observed in measurements of phosphoinositide hydrolysis. In the presence of NE, both second messenger responses were potentiated by the addition of CPA, whereas in the absence of NE, CPA had little or no effect on either second messenger. The stimulation and potentiation of both second messenger responses were enhanced in the presence of extracellular calcium, and in each case these effects were persistent over time. For either second messenger system the stimulation by NE and the potentiations by CPA appeared to utilize separate mechanisms as evidenced by the fact that the potentiations by CPA were selectively antagonized by a cAMP analogue or by pertussis toxin, whereas the stimulations by NE were essentially unaffected by these agents. Inhibition of phospholipase A 2 (PLA 2) also blocked the potentiation of PLC by CPA, without affecting NE-stimulated phosphoinositide hydrolysis. Furthermore, in the presence of CPA, the exogenous administration of PLA 2 was found to stimulate phosphoinositide hydrolysis in these cells. These data are consistent with a hypothesis whereby the apparent potentiation of NE-stimulated phosphoinositide hydrolysis by CPA is actually due to the stimulation by CPA of a second pathway of phospholipase C activity which is additive to that of NE. The activation of PLC and PLA 2 by NE produces phospholipid products which may play a permissive role in the pathway coupling adenosine A 1 receptors to these phospholipases. The formation of lysophosphatidic acid is suggested as one possible mediator of this permissive effect.

Effects of quisqualic acid analogs on metabotropic glutamate receptors coupled to phosphoinositide hydrolysis in rat hippocampus

l-Glutamic acid (L-Glu) and l-aspartic acid (L-Asp) activate several receptor subtypes, including metabotropic Glu receptors coupled to phosphoinositide (PI) hydrolysis. Quisqualic acid (Quis) is the most potent agonist of these receptors. There is evidence that activation of these receptors may cause a long lasting sensitization of neurons to depolarization, a phenomenon called the Quis effect. The purpose of the current studies was to use Quis analogs and the recently identified metabotropic receptor antagonist, (+)-α-methyl-4-carboxy-phenylglycine((+)-MCPG), to define the structural properties required for interaction with the metabotropic receptors coupled to PI hydrolysis and to determine if the Quis effect is mediated by these receptors. The effects of Quis analogs on PI hydrolysis were studied in the absence or presence of the metabotropic receptor-specific agonist 1 SR,3 RS-1-amino-1,3-cyclopentanedicarboxylic acid (1 SR,3 RS-ACPD) in neonatal rat hippocampus. Some of the compounds that induce the Quis effect also stimulate PI hydrolysis, including Quis itself and 9 (homoquisqualic acid). Not all of the Quis analogs that stimulate PI hydrolysis, however, induce the Quis effect, including 7A (EC 50 = 750 ± 150 μM) and ( RS)-4-bromohomoibotenic acid (BrHI) (EC 50 = 130 ± 40 μM). Although (+)-MCPG blocked PI hydrolysis stimulated by Quis (IC 50 = 370 ± 70 μM), it had no effect on the induction of the Quis effect. Other Quis analogs did not stimulate PI hydrolysis but rather blocked the effects of 1 SR,3 RS-ACPD. The IC 50 values were 240 ± 70 μM for 2, 250 ± 90 μM for 3, and 640 ± 200 μM for 4. Data for inhibition by 2 and 3 were consistent with non-competitive mechanisms of action. These studies provide new information about the structural features of Quis required for interaction with metabotropic receptors coupled to PI hydrolysis and provide evidence that the Quis effect is not mediated by (+)-MCPG sensitive subtypes of these receptors.

Multifunctional phosphatidic acid signaling in mammary epithelial cells: Stimulation of phosphoinositide hydrolysis and conversion to diglyceride

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide-specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen-coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with 3H-myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E2 affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with 3H-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride.