Stimulation Of Phosphoinositide Turnover Research Articles

FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

Background and ObjectiveMuscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).MethodsVariants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells.ResultsThe variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.ConclusionThe M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M1 mAChR activation.

Serotonin-2 (5-HT2) Receptor-Mediated Signal Transduction in Human Ciliary Muscle Cells: Role in Ocular Hypotension

The aim of this study was to characterize the serotonin (5-hydroxytryptamine; 5-HT) receptors that mediate phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) mobilization in isolated cells of human ciliary muscle (h-CM) from multiple donors using a variety of agonists and antagonists. An additional aim was to visualize the mRNAs and receptor binding sites for 5-HT2 receptors in human ciliary body (h-CB), CM, and other tissues by reverse transcriptase polymerase chain reaction and quantitative autoradiography techniques, respectively, and to correlate with ocular hypotensive activity of such compounds. CBs isolated from several donor eyes revealed the presence of 5-HT(2A2C) receptor mRNAs. [3H]-5-HT and [3H]-ketanserin autoradiography on sections of human eyes revealed a high density of 5-HT2 receptor binding sites in the iris, ciliary epithelium, and longitudinal CM. In isolated h-CM cells, the agonists alpha-methyl-5-HT (EC50=63+/-17 nM), 5-HT (EC50=85+/-16 nM), (R)-DOI (EC50=165+/-47 nM), and 5-methoxy alpha-methyl tryptamine (EC50=1200+/-270 nM) differentially stimulated PI turnover. These agonists also mobilized [Ca2+]i in h-CM cells with the following potencies (EC50s): 5-methoxy-tryptamine=42+/-11 nM; alpha-methyl-5-HT=36+/-11 nM; (R)-DOI=120 nM; 5-HT=130+/-36 nM; MK-212=470 nM; mCPP>1 microM; BW723C86=1766 nM. The agonist-induced [Ca2+]i mobilization in h-CM cells was potently blocked by the 5-HT2A-selective antagonist M-100907 (IC50=1.2+/-0.4 nM) but less potently by the antagonists for 5-HT2B (RS-127445, IC50>10 microM) and 5-HT2C (RS-102221, IC50=5.8+/-2.3 microM) receptors. In conclusion, h-CB, h-CM, and CM cells express mRNAs and proteins for 5-HT2 receptor subtypes, of which the predominant functionally active subtype is the 5-HT2A receptor, as defined by agonist and antagonist activities. These receptors may be responsible for mediating the intraocular pressure reduction observed in recent literature with a number of 5-HT2 agonists, such as (R)-DOI, alpha-methyl-5HT, and AL-34662.

Phosphoinositide turnover associated with synaptic transmission.

Although pharmacological stimulation of a wide variety of transmitter receptors triggers phosphoinositide (PI) turnover, little is known about the type of synaptic activity required to activate this system. To investigate this question, we have used primary cultures of embryonic cortical neurons, which develop functional glutamate and GABA synapses during maturation in vitro. Mature cultures display spontaneous synaptic activity that is totally suppressed by tetrodotoxin (TTX). PI turnover, assayed by the lithium-sensitive accumulation of [3H]CDP-diacylglycerol, was readily detected under basal conditions and was abolished by TTX. Increased excitatory synaptic activity induced by picrotoxin, an antagonist of GABAA receptor-mediated inhibition, further stimulated PI turnover. Similar results were obtained when PI turnover was assayed using [3H]inositol labeling. With either assay, the magnitude of synaptically induced PI turnover was comparable to maximal responses produced by muscarinic receptor stimulation. Although a component of the spontaneous synaptic currents is sensitive to N-methyl-D-aspartate (NMDA)-preferring glutamate receptor antagonists, blockade of NMDA receptors did not affect PI turnover associated with synaptic transmission. To assess the time course of synaptically mediated PI turnover, the amplitude and duration of spontaneous synaptic currents were reduced by lowering the extracellular Ca2+ concentration from 2.25 to 0.5 mM, a maneuver that suppresses basal PI turnover. Increases in PI turnover were detected as early as 5 min following restoration of the extracellular Ca2+ concentration to 2.25 mM. Taken together, these findings indicate that activation of the PI system is associated with physiological levels of glutamatergic synaptic transmission.

Human Trabecular Meshwork Cells Express Functional Serotonin-2A (5HT2A) Receptors: Role in IOP Reduction

To apply a multidisciplinary approach to the identification and pharmacological characterization of the serotonin (5HT) receptors that mediate functional responses in human trabecular meshwork (h-TM) cells. To correlate in vitro findings with intraocular pressure (IOP) changes in conscious ocular hypertensive cynomolgus monkeys. Documented methods were used, including reverse transcription-polymerase chain reaction (RT-PCR), phosphoinositide (PI) turnover, and intracellular Ca2+ ([Ca2+]i) mobilization. IOP was measured using standard applanation pneumatonometry. h-TM cells expressed robust mRNA signals for 5HT2A and 5HT2B receptors. 5HT and its analogues stimulated PI turnover and [Ca2+]i mobilization in h-TM cells from multiple donors (20/24 donors' TM cells responded). The agonist potencies (EC50) of compounds in mobilizing [Ca2+]i were (nM): 5-methoxy tryptamine, 8 +/- 4; (R)-DOI, 18 +/- 6; alpha-methyl-5HT, 22 +/- 3; 5HT, 40 +/- 7; 5-methoxy-dimethyl tryptamine, 64 +/- 27; and BW-723C86, 1213 +/- 210. These effects were potently blocked by the 5HT2A-receptor-selective antagonist, M-100907 (Ki = 1 +/- 0.3 nM), but weakly by antagonists of 5HT2B and 5HT2C receptors. Only 5HT2 receptor agonists such as (R)-DOI (300 microg lowered IOP 34.4% from baseline of 38.2 mm Hg; P < 0.001) and alpha-methyl-5HT (250 microg lowered IOP 30.8% from baseline of 41.8 mm Hg; P < 0.001) lowered IOP in ocular hypertensive cynomolgus monkeys. Strong signals for 5HT2A and 5HT2B receptor mRNAs were detected in h-TM cells. The receptors that coupled to PI hydrolysis and [Ca2+]i mobilization in h-TM cells were the 5HT2A receptor subtype, which also significantly lowered IOP in a primate model. These receptors may mediate the ocular hypotensive actions of 5HT2A agonists.

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla. 2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP(1)) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure. 3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [(3)H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET(B) receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP(1) accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET(A) receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET(B) receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP(1) accumulation. 4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET(A) receptor, while the latter through the activation of ET(B) receptor. These results support the role of endothelins in the regulation of adrenal medulla function.

Assay of Receptor‐Stimulated Phosphoinositide Turnover

The stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein-coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [(3)H]myo-inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal-to-noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion-exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)-trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [(3)H]myo-inositol, chromatography columns, tissue slices, and the IP(3)-binding protein.

Endothelin ETB receptor signaling in the median eminence and subfornical organ of the rat brain.

We investigated the effect of endothelins (ETs) on receptor-mediated phosphoinositides (PI) turnover in whole subfornical organ (SFO) and median eminence (ME). Consistent with the presence of a high density of binding sites in the SFO and the ME of the rat brain, our results show an increase in PI hydrolysis induced by ETs in each structure, in a dose-dependent manner and with similar ED50 values. In addition, IRL 1620, a selective ETB receptor agonist, increased the inositol monophosphate (InsP1) accumulation in the SFO and the ME in a similar degree as ETs. With the use of selective agonists and antagonists of both endothelin receptor subtypes, we characterized the receptor subtype involved in ET-induced phosphoinositide metabolism. The addition of two selective ETA receptor antagonists, BQ 123 or BQ 610, did not alter the ETs-induced increase in the PI metabolism. While, IRL 1620- and ET3-induced InsP1 accumulation was completely blocked by BQ 788, a selective ETB receptor antagonist, in both brain structures evaluated. Our results demonstrate that in the SFO and the ME of the rat brain, stimulation of phosphoinositide turnover constitutes one of the signaling pathways of ETs, and this action is mediated through ETB receptor activation. These results support the concept that endothelin could play a role in the regulation of brain functions.

Identification and Molecular Characterization of Two Closely Related G Protein-coupled Receptors Activated by Prokineticins/Endocrine Gland Vascular Endothelial Growth Factor

We previously described two mammalian secreted proteins, prokineticin 1 and prokineticin 2, that potently contract gastrointestinal smooth muscle. Prokineticin 1 has also been shown to promote angiogenesis by stimulating proliferation, migration, and fenestration of endocrine organ-derived endothelial cells. Here we report the cloning and characterization of two closely related G protein-coupled receptors as receptors for prokineticins. Expression of prokineticin receptors in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant prokineticins. Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of prokineticins on smooth muscle contraction and angiogenesis. mRNA expression analysis reveals that prokineticin receptors are expressed in gastrointestinal organs, endocrine glands, and other tissues.

An endogenous Na+, K+-ATPase inhibitor enhances phosphoinositide hydrolysis in neonatal but not in adult rat brain cortex.

The effect of an endogenous Na+, K+-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in rat brain cortical prisms and compared with that of ouabain. As already shown for ouabain, a transient effect was obtained with endobain E; maximal accumulation of inositol phosphates induced by endobain E was 604 +/- 138% and 186 +/- 48% of basal values in neonatal and adult rats, respectively. The concentration-response plot for the interaction between endobain E and phosphoinositide turnover differed from that of ouabain, thus suggesting the involvement of distinct mechanisms. In the presence of endobain E plus ouabain at saturating concentrations, no additive effect was recorded, suggesting that both substances share at least a common step in their activation mechanism of inositol phosphates metabolism or that they enhance phosphatidylinositol 4,5-biphosphate breakdown from the same membrane precursor pool, until its exhaustion. Experiments with benzamil, a potent blocker of Na+/Ca2+ exchanger, showed that it partially and dose-dependently inhibited endobain E effect. These results indicate that the endogenous Na+, K+-ATPase inhibitor endobain E, like ouabain, is able to stimulate phosphoinositide turnover transiently during postnatal brain development.

Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3

An increase of intracellular cAMP mediated by prostaglandin E2 (PGE2) has been shown to delay spontaneous apoptosis of neutrophils. It has been demonstrated that a selective agonist for PGE2 receptor subtype 3 (the EP3 receptor) is capable of decreasing cAMP and stimulating phosphoinositide turnover in various types of cells. We investigated the effect of a selective EP3 receptor agonist, ONO-AE-248, on neutrophil viability. ONO-AE-248 rapidly caused a unique form of neutrophil death. The agonist primarily induced morphological changes of the nucleus, including fusion of the lobules, decreased compactness of the chromatin, and blebbing and rupture of the nuclear membrane. This was followed by an increase of plasma membrane permeability and cell lysis. During these processes, neither apoptotic changes such as nuclear condensation, DNA fragmentation, and expression of phospholipid phosphatidylserine on the plasma membrane nor necrotic changes such as chromatin clumping and organelle destruction were apparent in the treated neutrophils. The fatal effect of the agonist night be specific for neutrophils because it failed to promote the rapid death of other types of cells. Although activation of neutrophils by ONO-AE-248 was not evident, experiments using metabolic inhibitors demonstrated that the agonist caused neutrophil death via the activation of protein kinase C in the presence of intracellular ATP. These findings indicated that EP3 receptor-mediated signals might promote a novel form of neutrophil death, which differs from typical apoptosis or necrosis.

Assay of Receptor‐Stimulated Phosphoinositide Turnover

AbstractThe stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein‐coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [3H]myo‐inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal‐to‐noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion‐exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)‐trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [3H]myo‐inositol, chromatography columns, tissue slices, and the IP3‐binding protein.

Metabotropic Glutamate Receptor-Mediated Hippocampal Phosphoinositide Turnover Is Blunted in Spatial Learning-Impaired Aged Rats

Maximal phosphoinositide (PI) turnover was examined in the hippocampus of young and aged Long-Evans rats that were behaviorally characterized for spatial learning in the Morris water maze. The type 1 metabotropic glutamate receptor (mGluR) agonist 1S,3R ACPD was used to stimulate PI turnover and to determine the E(MAX) for each rat. Protein levels in hippocampus for type 1 mGluRs, Galphaq11, and phospholipase Cbeta-1 (PLCbeta-1) were also measured by quantitative Western blotting. The results show that PI turnover mediated by the mGluRs was blunted in the aged rats. The magnitude of the decrement in PI turnover was also significantly correlated with age-related spatial memory decline. The decrease in mGluR-mediated PI turnover occurred without changes in the protein level of either the mGluRs or the G-protein coupled to those receptors, Galphaq11. A significant decrease in the immunoreactivity of PLCbeta-1, however, was observed in the hippocampus of aged rats; PLCbeta-1 immunoreactivity was significantly correlated with spatial learning only when the young and aged rats were considered together. The decrement in mGluR-mediated signal transduction in the hippocampus that is related to cognitive impairment in aging may be attributable, at least in part, to a deficiency in the enzyme PLCbeta-1. That deficiency may also contribute to a blunted response in muscarinic stimulation of hippocampal PI turnover that we previously found in this same study population. An age-related alteration in this signal transduction system may provide a functional basis for cognitive decline independent of any loss of neurons in the hippocampus.

Endothelin-1 Stimulates Phosphoinositide Hydrolysis in the Rat Pineal Gland

The presence of functional endothelin receptors and their signal transduction mechanism has not been determined so far in the pineal gland. We examined the effect of endothelin-1 (ET-1) on phosphoinositide turnover in whole pineal gland. Endothelin-1 increased monophosphate accumulation in a dose-dependent manner. The phosphoinositide (PI) response elicited by ET-1 was dependent on the presence of extracellular Ca ++ since its chelation resulted in a marked decrease in ET-1-stimulated InsP 1 accumulation. On the contrary, phosphoinositide hydrolysis was not changed by the calcium blocker amlodipine. ET-1 induced PI breakdown was inhibited by neomycin, an inhibitor of phospholipase C. However, mastoparan 7, a G protein activator via Gi/Go s timulation, did not alter ET-1-induced InsP 1 accumulation. Our data indicate that stimulation of PI turnover constitutes one of the signaling pathways of ET in rat pineal gland through the stimulation of a receptor-coupled phospholipase C. And they demonstrate, for the first time, the presence of functional binding sites for endothelin in the pineal gland.

Mastoparan induces an increase in cytosolic calcium ion concentration and subsequent activation of protein kinases in tobacco suspension culture cells

Mastoparan induced a transient elevation of cytosolic free Ca 2+ concentration ([Ca 2+] cyt) in tobacco suspension culture cells. The mastoparan-induced [Ca 2+] cyt elevation was inhibited by 8-( N, N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl and neomycin but not by depletion of extracellular Ca 2+, suggesting that the elevation was the result of Ca 2+ release from the intracellular stores caused by stimulation of phosphoinositide turnover. Hydrogen peroxide which has been shown to induce an oxidative burst in soybean cells by mastoparan treatment [L. Legendre, P.F. Heinstein, P.S. Low, Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells, J. Biol. Chem., 267 (1992) 20140–20147], also induced a transient [Ca 2+] cyt elevation in the tobacco cells. However, mastoparan did not induce an oxidative burst in the tobacco cells. Activation of a 50, a 75 and a 80 kDa protein kinases after the mastoparan-induced [Ca 2+] cyt elevation was shown by an in-gel protein kinase assay. This activation was inhibited by neomycin, suggesting that the [Ca 2+] cyt elevation is necessary for the mastoparan-induced activation of the protein kinases. The activation was inhibited also by pretreatment with staurosporine and was sustained by pretreatment with calyculin A, suggesting that the protein kinase activity is regulated by protein phosphorylation/dephosphorylation. The present report shows that mastoparan induces an increase in [Ca 2+] cyt without oxidative burst and subsequent activation of protein kinases in tobacco cells.

Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase-1 release from human corneal epithelial cells.

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B2-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells. 2. The aims of the present studies were to demonstrate the specific binding of [3H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+-mobilization ([Ca2+]i), cell proliferation (via [3H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E2 (PGE2). 3. Specific [3H]-BK binding comprised 83 +/- 2% of the total binding, and was of high affinity (Kd = 1.66 +/- 0.52 nM, n = 5), saturable (Bmax = 640 +/- 154 fmol g(-1) wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]BK; icatibant): Ki = 0.17 +/- 0.07 nM; BK: Ki = 1.0 +/- 0.11 nM; [Tyr8]-BK: Ki = 12.9 +/- 2.3 nM; [des-Arg9]-BK: Ki > 9,200 nM (all n = 3-5)). 4. BK potently stimulated PI turnover (EC50 = 2.3 +/- 0.3 nM; n = 7) and [Ca2+]i mobilization (EC50 = 8-20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM-10 microM Hoe-140, a selective B2-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione) (IC50 = 3.0 +/- 1.6 microM). BK-induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine. 5. BK (0.1 nM-10 microM) significantly (P<0.05-0.001) stimulated [3H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1alpha (IL-1alpha; 10 ng ml(-1)) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM-10 microM) was without effect. 6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 microg ml(-1)) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE2 from CEPI cells, BK (0.1 nM-10 microM) was without any significant effect under these conditions. 7. In conclusion, these data indicate that the CEPI cells express high-affinity [3H]-BK binding sites representing B2-subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

Chronic dosing with 1-aminocyclopropanecarboxylic acid, a glycine partial agonist, modulates NMDA inhibition of muscarinic-coupled PI hydrolysis in rat cortical slices.

Chronic dosing with the glycine partial NMDA agonist, 1-aminocyclopropanecarboxylic acid (ACPC) elicited an altered allosteric regulation of cortical NMDA receptor binding. The present study hypothesized that these allosteric receptor binding changes would be manifest as pharmacologically functional reductions in NMDA receptor activity following chronic ACPC dosing. NMDA inhibition of carbachol-induced phosphoinositide (PI) hydrolysis was used as a functional assay to assess NMDA receptor function in rat cerebral cortex. NMDA inhibition of stimulated PI turnover was similar in naive (46% +/- 4.5%; mean +/- SE; n = 34) and ACPC dosed rats (39% +/- 2.3%; n = 34). While ACPC reversed NMDA's inhibitory effects in naive rats (80% +/- 13%; n = 9), it was ineffective (P < 0.05) in ACPC pretreated rats (48% +/- 9.8%; n = 9). In contrast, the NMDA antagonists, MK-801 (ion channel), 7-chlorokynurenic acid (glycine site) and AP-7 (glutamate site), effectively reversed NMDA's inhibitory effects in naive and ACPC treated rats. The potency of these antagonists were unaltered by prior ACPC dosing. Thus, chronic ACPC therapy does not alter the functioning of the NMDA ion channel or glutamate receptor sites, but elicits functional tolerance to ACPC's actions in the cortical NMDA complex.

Dihydrobenzofuran analogues of hallucinogens. 4. Mescaline derivatives.

Dihydrobenzofuran and tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in the prototypical hallucinogen, mescaline (1). Thus, 4-(2-aminoethyl)-6,7-dimethoxy-2,3-dihydrobenzofuran hydrochloride (8) and 1-(8-methoxy-2,3,5,6-tetrahydrobenzo[1,2-b:5,4-b']difuran-4-yl)-2- aminoethane hydrochloride (9) were prepared and evaluated along with 1 for activity in the two-lever drug discrimination (DD) paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg). Also, 1, 8, and 9 were assayed for their ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors and [3H]8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, these compounds were evaluated for their ability to compete for agonist and antagonist binding to cells expressing cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Finally, agonist efficacy was assessed by measurement of phosphoinositide hydrolysis in NIH 3T3 cells expressing the rat 5-HT2A or 5-HT2C receptors. Although 1 fully substituted for LSD in the DD assays (ED50 = 33.5 mumol/kg), neither 8 nor 9 substituted for LSD, with just 50% of the rats administered 8 selecting the drug lever, and only 29% of the rats administered 9 selecting the drug lever. All of the test compounds had micromolar affinity for the 5-HT1A and 5-HT2A receptors in rat brain homogenate. Curiously, the rank order of affinities of the compounds at 5-HT2A sites was opposite their order of potency in the behavioral assay. An evaluation for ability to stimulate phosphoinositide turnover as a measure of functional efficacy revealed that all the compounds were of approximately equal efficacy to serotonin in 5-HT2C receptors. At 5-HT2A receptors, however, 8 and 9 were significantly less efficacious, eliciting only 61 and 45%, respectively, of the maximal response. These results are consistent with the proposed mechanism of action for phenethylamine hallucinogens, that such compounds must be full agonists at the 5-HT2A receptor subtype. In contrast to the 2,5-dimethoxy-substituted phenethylamines, where rigidification of the methoxy groups had no deleterious effect on activity, the loss of activity in the 3,4,5-trioxygenated mescaline analogues may suggest that the 3 and 5 methoxy groups must remain conformationally mobile to enable receptor activation.

Effect of Chronic Ethanol Exposure on Myocardial Phosphoinositide Turnover

The effect of chronic ethanol consumption (2 months) on atrial contractility and the myocardial phosphoinositide signaling system was examined in rat heart. Two months of ethanol consumption was not associated with changes in heart weight-to-body weight ratios; however, developed twitch tension was significantly lower in the ethanol atria compared with the control atria. Cytosolic and membrane-associated phospholipase C activity in atrial and ventricular tissue was measured and ethanol consumption was only associated with changes in ventricular cytosolic phospholipase C activity. When examining alpha(1)-adrenergic stimulated phosphoinositide turnover in [3H]inositol radiolabeled left atria, no differences in phenylephrine (10 microM)-stimulated inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate were found between groups before or at various times after the addition of phenylephrine. It is concluded that short-term ethanol consumption is associated with depressed contractile function, but not the development of hypertrophy or changes in alpha(1)-adrenoreceptor-stimulated phosphoinositide turnover.

Discovery of TBC11251, a potent, long acting, orally active endothelin receptor-A selective antagonist.

Previously we reported the discovery of amidothiophenesulfonamides as endothelin receptor-A antagonists with high potency and selectivity. Replacement of an amide group in this class of compounds with an acetyl group maintained the in vitro binding affinity and in vivo activity while providing a compound with oral bioavailability and longer duration of action. The optimal compound discovered during these studies, 15q (TBC11251), binds competitively to human ETA receptors with a Ki of 0.43 +/- 0.03 nM and an IC50 of 1.4 nM (IC50 for ETB = 9800 nM). This compound inhibits ET-1-induced stimulation of phosphoinositide turnover with a Ki of 0.686 nM and a pA2 of 8.0. The compound has a serum half-life in the rat and the dog of 6-7 h and 60-100% oral bioavailability. This compound is one of the most selective ETA antagonists reported and therefore is suitable for additional pharmacological and clinical investigation of the role of ETA receptors in diseases.

Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover.

The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.