Phosphoinositide Hydrolysis In Cells Research Articles

Cyclobutane quisqualic acid analogues as selective mGluR5a metabotropic glutamic acid receptor ligands.

The conformationally constrained cyclobutane analogues of quisqualic acid (Z)- and (E)-1-amino-3-[2'-(3',5'-dioxo-1',2', 4'-oxadiazolidinyl)]cyclobutane-1-carboxylic acid, compounds 2 and 3, respectively, were synthesized. Both 2 and 3 stimulated phosphoinositide (PI) hydrolysis in the hippocampus with EC50 values of 18 +/- 6 and 53 +/- 19 microM, respectively. Neither analogue stimulated PI hydrolysis in the cerebellum. The effects of 2 and 3 were also examined in BHK cells which expressed either mGluR1a or mGluR5a receptors. Compounds 2 and 3 stimulated PI hydrolysis in cells expressing mGluR5a but not in those cells expressing mGluR1a. The EC50 value for 2 was 11 +/- 4 microM, while that for 3 was 49 +/- 25 microM. Both 2 and 3 did not show any significant effect on cells expressing the mGluR2 and mGluR4a receptors. In addition, neither compound blocked [3H]glutamic acid uptake into synaptosomal membranes, and neither compound was able to produce the QUIS effect as does quisqualic acid. This pharmacological profile indicates that 2 and 3 are selective ligands for the mGluR5a metabotropic glutamic acid receptor.

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 short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol.

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, 3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in 3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18 +/- 4 pg/islet/min, peak first and second phase responses now averaged 422 +/- 61 and 1016 +/- 156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20-24 h in CMRL-1066 medium attenuated all measured responses. The increases in 3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone 68 +/- 9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358 +/- 85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.

Constitutively active 5-hydroxytryptamine2C receptors reveal novel inverse agonist activity of receptor ligands.

5-HT2C receptor antagonists, such as mianserin and mesulergine, exhibit negative intrinsic activity, defined as a decrease in agonist-independent, receptor-mediated, phosphoinositide hydrolysis in cells transfected with the 5-HT2C receptor cDNA. These drugs are classified as inverse agonists. Guanine nucleotides reciprocally modulate the binding of an agonist and inverse agonist, suggesting that an inverse agonist binds preferentially to the G protein-uncoupled form of the 5-HT2C receptor. Another 5-HT2C receptor antagonist, 2-bromolysergic acid diethylamide, functions as a neutral antagonist with no intrinsic activity, but is able to block both agonist and inverse agonist. Chronic treatment of choroid plexus cells with an inverse agonist, but not with the neutral antagonist, causes 5-HT2C receptor down-regulation, suggesting that the biological effects of 5-HT2C receptor antagonists are not solely due to antagonism of endogenous agonist. These results provide evidence that constitutively active 5-HT2C receptors are biologically significant. The functionally distinct properties of inverse agonists and neutral antagonists may elucidate the mechanisms controlling basal receptor activity states and lead to novel approaches in the development of therapeutic agents.

Regulation of arachidonic acid release in mouse peritoneal macrophages. The role of extracellular calcium and protein kinase C.

Primary cultures of [3H]arachidonic acid-prelabeled mouse peritoneal macrophages were stimulated with the physiologic agonists zymosan and Con A. The cells released a large quantity of labeled compounds into the extracellular medium. When the cells were preincubated for 10 min with PMA at concentrations from 10 to 1000 ng/ml, zymosan- and Con A-stimulated compound release was greatly enhanced. PMA also potentiated arachidonic acid release when cells were stimulated with the calcium ionophore A23187. However, under the same conditions, PMA treatment blocked agonist- and ionophore-mediated phosphoinositide hydrolysis in cells prelabeled with [3H]inositol. Thus, PMA treatment appears to have dissociated agonist-induced arachidonic acid liberation (index of phospholipase A2) from phosphoinositide hydrolysis (index of phospholipase C), suggesting that these two coupling processes can occur in a parallel and independent manner in mouse peritoneal macrophages. Furthermore, the ligand-induced arachidonic acid release from PMA-treated macrophages was shown to be directly dependent on the extracellular calcium concentration. Considering that in PMA-pretreated cells, receptor-mediated phosphoinositide breakdown and intracellular calcium mobilization were abolished, our data suggest that the extracellular calcium influx that takes place after receptor-ligand interaction may be a required event for arachidonic acid mobilization in mouse peritoneal macrophages.