Activation Of Phosphoinositide Hydrolysis Research Articles

Assessment of jaw movements by magnetic sensor in relation to topographies of orofacial behaviour in freely moving rats: Studies with the dopamine D 1-like receptor agonists SKF 83822 vs SKF 83959

This study applies new magnetic sensor-electromyographic technology for recording jaw movements in freely moving rats to analyse topographies of orofacial movement that occur in association with individual elements of behaviour under challenge with two dopamine D 1-like receptor agonists, SKF 83822 ([ R/S]-6-chloro-7, 8-dihydroxy-3-allyl-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-1 H-3-benzazepine) and SKF 83959([ R/S]-3-methyl-6-chloro-7, 8-dihydroxy-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-1 H-3-benzazepine). Grooming of the snout/face involved primarily dominant-mouth opening jaw movements with small activation of digastric muscles; subsequent grooming of the flank/trunk was characterised by repetitive, uniform jaw movements with small activation of digastric and masseter muscles. In contrast, grooming of the fingers and tail typically involved high-frequency jaw movements with variable vertical jaw movements and/or strong activation of masseter muscles. Vacuous chewing involved two distinct patterns of jaw movements: a dominant-closing pattern, with strong activation of masseter muscles, and a dominant-opening pattern, with slight activation of masseter muscles. SKF 83822 stimulates dopamine D 1-like receptors and activates adenylate cyclase but not phosphoinositide hydrolysis, while SKF 83959 stimulates dopamine D 1-like receptors and activates phosphoinositide hydrolysis but not adenylate cyclase. These agonists exerted differential effects on jaw movements, as SKF 83959 induced more jaw movements per episode of syntactic grooming than SKF 83822, while SKF 83822 induced more jaw movements during non-syntactic grooming than SKF 83959. Magnetic sensor technology in freely moving animals resolved distinct topographies of orofacial movement and informs on their relationship to other behaviours in the rodent repertoire and to dopamine D 1-like receptor function.

Sediment and phosphorus removal from simulated storm runoff with compost filter socks and silt fence

Alanine scanning mutagenesis of B-B-X-X-B motifis (where B is a basic residue and X is any nonbasic residue) in m1 muscarinic acetylcholine receptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G protein-coupled receptors and has been implicated in G protein activation. The KKAAR365 motif, located at the carboxyl-terminal third intracellular loop of m1 receptors, was mutated to AAAAA365, thereby generating a triple-substitution mutant devoid of ability to stimulate either phosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a triple-alanine substitution of the KRTPR140 motif in the carboxyl-terminal second intracellular loop, yielding mutant AATPA140, had no effect on receptor coupling to the two independent second messenger pathways. Analysis of a series of single- and double-substitution mutants demonstrate that all three basic residues of the KKAAR365 motif participate in efficient m1 receptor coupling. The presence of second and third basic residues in this motif was absolutely critical for full agonist recognition of a high and low affinity state of the receptor. Mutation of either Lys362 or Lys365, but not-Lys361, abolished guanine nucleotide-dependent conversion of agonist affinity states and correlated with an inability of full agonists to fully activate PI hydrolysis. The different combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR365 motif, for efficient receptor coupling. This residue cannot facilitate full agonist-stimulated Pl hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both necessary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial ability to couple PI hydrolysis after full agonist stimulation. Therefore, Lys365 seems to function in a hierarchal (interdependent) manner with nearby basic residues, whereas Lys361 and Lys362 can act independent of surrounding basic residues to facilitate partial m1 receptor coupling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.

Activation and Regulation of Platelet-Activating Factor Receptor: Role of Gi and Gq in Receptor-Mediated Chemotactic, Cytotoxic, and Cross-Regulatory Signals

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.

Lysophosphatidic acid induces hypertrophy of neonatal cardiac myocytes via activation of G i and Rho

The effect of the lysophospholipid, lysophosphatidic acid (LPA), on signaling and hypertrophy of neonatal rat ventricular cardiomyocytes was examined. Myocytes express mRNA for all three G-protein-coupled LPA receptor subtypes (LPA 1/Edg-2, LPA 2/Edg-4, and LPA 3/Edg-7) as indicated by RT-PCR analysis. LPA inhibits isoproterenol-stimulated cyclic AMP accumulation with an IC 50 ~40 nM and promotes phosphorylation of ERK-1/2. LPA also elicits a small, slow onset, and activation of phosphoinositide hydrolysis with EC 50 ~400 nM, and stimulates a marked increase in the extent of Rho activation. Longer-term treatment with LPA induces a hypertrophic response in myocytes as indicated by increases in cell size, actin organization, ANF staining of the perinuclear region and activation of ANF promoter–luciferase gene expression. Pretreatment of myocytes with pertussis toxin (PTX) not only blocks the capacity of LPA to inhibit cyclic AMP formation and stimulate ERK phosphorylation, but also inhibits hypertrophic changes in cell morphology and ANF–luciferase gene expression. Neither phospholipase C nor Rho activation is PTX sensitive. The hypertrophic effects of LPA on myocytes are also inhibited by treatment with C3 exoenzyme or by transfection of plasmids expressing either C3 exoenzyme or dominant-negative Rho to block Rho function. Inhibition of ERK activation with PD98059 blocks LPA-induced hypertrophy while inhibitors of phospholipase C (U73122), PKC (GF109203X), or p38MAPK (SB203580) do not. These data suggest that LPA induces cardiomyocyte hypertrophy via a pathway different from the conventional G q pathway utilized by phenylephrine, endothelin, and PGF2α and involving activation of a PTX-sensitive G i/ERK pathway in conjunction with activation of Rho-mediated signals.

Differential modes of agonist binding to 5-hydroxytryptamine(2A) serotonin receptors revealed by mutation and molecular modeling of conserved residues in transmembrane region 5.

Site-directed mutagenesis and molecular modeling were used to investigate the molecular interactions involved in ligand binding to, and activation of, the rat 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin (5-HT) receptor. Based on previous modeling studies utilizing molecular mechanics energy calculations and molecular dynamics simulations, four sites (S239[5.43], F240[5.44], F243[5.47], and F244[5.48]) in transmembrane region V were selected, each predicted to contribute to agonist and/or antagonist binding. The F243A mutation increased the affinity of (+/-)4-iodo-2, 5-dimethoxyphenylisopropylamine, decreased the binding of alpha-methyl-5HT, N-omega-methyl-5HT, ketanserin, ritanserin, and spiperone and had no effect on the binding of 5-HT and 5-methyl-N, N-dimethyltryptamine. The F240A mutant had no effect on the binding of any of the ligands tested, whereas F244A caused an agonist-specific decrease in binding affinity (3- to 10-fold). S239A caused a 6- to 13-fold decrease in tryptamine-binding affinity and a 5-fold increase in affinity of 4-iodo-2, 5-dimethoxyphenylisopropylamine. A subset of the agonists used in binding studies were used to determine the efficacies and potencies of these mutants to activate phosphoinositide hydrolysis. The F243A and F244A mutations reduced agonist stimulated phosphoinositide hydrolysis, whereas the S239A and F240A mutations had no effect. There was little correlation between agonist binding and second messenger production. Furthermore, molecular dynamics simulations, considering these data, produced ligand-bound structures utilizing substantially different bonding interactions even among structurally similar ligands (differing by as little as one methyl group). Taken together, these results suggest that relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT(2A) receptor activation. Thus, our finding may have major implications for the future and feasibility of receptor structure-based drug design.

Characterization of the Ca2+ Response Mediated by Activation of β-Adrenoceptors in Rat Submandibular Ducts

The Ca2+ signaling mediated by activation of beta-adrenoceptors was studied in a purified preparation of ducts from rat submandibular glands. At concentrations above 1 nM, isoproterenol (ISO) caused a small but significant increase in cytosolic Ca2+ concentration ([Ca2+]i). The ISO-induced increase in [Ca2+]i was completely inhibited by the beta-adrenoceptor antagonist propranolol but not by the alpha-adrenoceptor antagonist phentolamine. Forskolin was able to mimic the Ca2+ response to ISO. These results suggest that the ISO-induced increase in [Ca2+]i in rat submandibular ducts is mediated by an accumulation of cAMP resulting from activation of beta-adrenoceptors. In the absence of extracellular Ca2+, ISO or forskolin caused a transient increase in [Ca2+]i, indicating Ca2+ mobilization from intracellular Ca2+ stores. Further, stimulation with ISO failed to mobilize Ca2+ after the depletion of intracellular Ca2+ stores by phenylephrine or carbachol, suggesting that the cAMP-mediated increase in [Ca2+]i is due to a Ca2+ release from inositol trisphosphate (IP3)-sensitive Ca2+ stores. As ISO did not stimulate a detectable production of IP3, the cAMP-mediated Ca2+ mobilization may be evoked by a mechanism different from activation of phosphoinositide hydrolysis.

Effects of varying the expression level of recombinant human mGlu1alpha receptors on the pharmacological properties of agonists and antagonists.

1. Different expression levels of the human type 1alpha metabotropic glutamate (mGlu1alpha) receptor were obtained in transfected Chinese hamster ovary cells using an isopropyl beta-D-thiogalactopyranoside (IPTG) inducible system. Expression of mGlu1alpha receptors could not be detected using immunoblotting or immunocytochemical approaches in non-induced cells, however, controlled expression could be induced following IPTG addition in a time- and concentration-dependent manner. 2. In induced cells (100 microM IPTG, 20 h) the agonists L-quisqualate or 1-aminocyclopentane-1S,3R-dicarboxylic acid stimulated large increases in [3H]-inositol (poly)phosphate (in the presence of Li+) and inositol, 1,4,5-trisphosphate levels. 3. Induction with 1-100 microM IPTG allowed the receptor density to be increased incrementally and this not only resulted in an increase in the maximum response to L-quisqualate, 1-aminocyclopentane-1S,3R-dicarboxylic acid and (S)-3,5-dihydroxy-phenylglycine, but also in an increase in the respective potencies of each agent to activate phosphoinositide hydrolysis. 4. The intrinsic activity of the partial agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid dramatically increased with increasing receptor expression. 5. The activities of the competitive mGlu1alpha receptor antagonists (S)-alpha-methyl-4-carboxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine for inhibition of the effects of L-quisqualate or (S)-3,5-dihydroxyphenylglycine were found to be independent of the receptor expression level. 6. When the mGlu1alpha receptor was expressed at very high levels, no evidence for receptor constitutive activity could be detected, and none of the antagonists tested revealed either any intrinsic activity or negative efficacy. 7. These data demonstrate that both the potency and efficacy of mGlu1alpha receptor agonists are influenced by expression level, whilst mGlu1alpha receptor antagonist activities are independent of expression level.

Digital imaging of intracellular Ca 2+ signaling in rat parotid acinar cells

Utilizing digital imaging microscopy, the receptor-mediated changes in cytosolic Ca 2+ concentration ([Ca 2+] i) were studied in fura-2-loaded rat parotid acinar cells. The increase in [Ca 2+] i induced by carbachol was initiated in the apical pole of the acinar cells and then spread as a Ca 2+ wave toward the basolateral region. A similar polarization of Ca 2+ signal was observed when the acinar cells were stimulated with substance P or phenylephrine. As the microsomal Ca 2+-ATPase inhibitor thapsigargin did not produce a Ca 2+ wave, activation of phosphoinositide hydrolysis is probably essential to trigger the Ca 2+ wave. Stimulation with 1 μM isoproterenol, a concentration which causes the maximum release of amylase, had no effect on [Ca 2+] i. Extracellular ATP (0.5 mM) induced a homogeneous increase in [Ca 2+] i throughout the cells in the presence of extracellular Ca 2+ but did not change [Ca 2+] i in the absence of extracellular Ca 2+, indicating that the ATP-induced rise in [Ca 2+] i is due to Ca 2+ entry.

Suppression of Thyrotropin Receptor-G Protein-Phospholipase C Coupling by Activation of Protein Kinase C in Thyroid Carcinoma Cells*

In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cbeta (PLCbeta)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCbeta in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCbeta signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.

Immortalized hypothalamic neurons express metabotropic glutamate receptors positively coupled to cyclic AMP formation.

We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S,3R)-cyclopentanedicarboxylic acid (1S,3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GT1-7 cells stained positively for the subtypes mGluR1a, -1b and -5 (belonging to group I mGluR2/3 (group II) and mGluR7 (group III). Agonists of group I mGluRs, including 1S,3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S,3R-ACPD effectively released GnRH in confluent cultures. We then focused on group II and III mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonist which preferentially activate group II and III mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of cAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as beta-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a Gs-type of GTP-binding protein.

Muscarinic receptor heterogeneity in neonatal rat ventricular myocytes in culture.

Carbachol increased ventricular automaticity in a concentration-dependent fashion from a control rate of 72 +/- 5 (mean +/- SEM) to 86 +/- 4 beats per minute at 10(-4) M carbachol. Pirenzepine, an M1-selective antagonist, and AFDX 116, an M2-selective antagonist, both at 10(-7) M, did not block the carbachol-induced positive chronotropic response. In contrast, 10(-7) M HHSiD, an M3-selective antagonist, completely blocked the positive chronotropic effect of carbachol. Carbachol stimulated the accumulation of IP1 in a concentration-dependent manner at concentrations > or = 3 x 10(-6) M. AFDX 116 had no effect on carbachol-induced IP1 accumulation. HHSiD significantly inhibited IP1 accumulation at concentrations > or = 3 x 10(-8) M, while pirenzepine inhibited IP1 accumulation only at concentrations > or = 10(-5) M. McN A343 and methacholine, two muscarinic receptor agonists with minimal M2 activities, and carbachol did not alter basal cAMP concentration, but all three agonists significantly attenuated the increase in cAMP accumulation in response to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations > or = 10(-7) M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrations, the inhibitory effects of HHSiD and AFDX-116 were similar, while that of pirenzepine was much less. Pretreatment with pertussis toxin for 24 h did not prevent the carbachol-mediated positive chronotropic response or accumulation of IP1 but completely abolished the inhibition of isoproterenol-stimulated cAMP accumulation. These results indicate that (a) neonatal ventricular myocytes in culture have a heterogeneous population of muscarinic (M2 and M3) receptors, (b) the M3 receptor is coupled to pertussis toxin-sensitive and pertussis toxin-insensitive G proteins, (c) M3 receptor stimulation activates phosphoinositide hydrolysis and increases automaticity via a pertussis toxin-insensitive G protein-dependent pathway, and (d) both M2 and M3 receptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproterenol stimulation.

Pharmacological basis for functional selectivity of partial muscarinic receptor agonists

Muscarinic receptor agonists activate phosphoinositide hydrolysis and adenylate cyclase in Chinese hamster ovary cells transfected with cDNAs encoding the human muscarinic m1 and m 3 receptors. Whereas carbachol activates similarly both receptor subtypes, 4-[3-chlorophenyl-carbamoyloxy]-2-butynyltrimethyl ammonium chloride (McN-A-343) preferentially activates the m1 subtype over m3, in regard to both phosphoinositide hydrolysis and adenylate cyclase activity. On the other hand, oxotremorine activates phosphoinositide hydrolysis to a similar extent in both cell lines, but it activates preferentially adenylate cyclase in m1 versus m3 receptor expressing cells. Relative to carbachol, both McN-A-343 and oxotremorine activate preferentially phosphoinositide hydrolysis over adenylate cyclase in both cell lines. Prolonged incubation of cells with either carbachol, McN-A-343, or oxotremorine down-regulated the m1 receptors. This was accompanied by a parallel decrease in adenylate cyclase activity, whereas phosphoinositide hydrolysis remained relatively high. Inactivation of the receptors by alkylation with acetylethylcholine mustard, or by blocking with atropine, reduced carbachol-stimulated adenylate cyclase activity more effectively than carbachol-induced phosphoinositide hydrolysis in both m1 and m3 receptor expressing cells. These findings imply that the receptor reserve in these cell lines is greater for phosphoinositide hydrolysis response than for adenylate cyclase response. Yet, the receptor reserve for each of these responses is similar in both m1 and m3 receptor expressing cells. Since the binding affinities of McN-A-343 and of oxotremorine to m1 and m3 receptors are very similar, and since both cell lines contain similar amounts of spare receptors, we propose that the preferential activation of muscarinic m1 over m3 receptor by partial agonists is related to differences in the abilities of the two receptor subtypes to undergo conformational changes following agonist binding. This hypothesis is supported by results showing that the muscarinic m1 but not m3 receptor exhibits two affinity states in a competition binding assay.

Down-regulation of the G-proteins Gqα and G11α by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation

Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.

Type I, II, and III Inositol 1,4,5-Trisphosphate Receptors Are Unequally Susceptible to Down-regulation and Are Expressed in Markedly Different Proportions in Different Cell Types

The type I inositol 1,4,5-trisphosphate (InsP3) receptor can be rapidly depleted from cells during stimulation of phosphoinositide hydrolysis because its degradation is accelerated (Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol. Chem. 269, 7963-7969). The present study examines the regulatory properties of type II and III InsP3 receptors. Initially, the relative abundance of InsP3 receptors was defined in a range of cell types by quantitative immunoblotting. These studies showed that the proportions in which type I, II, and III InsP3 receptors are expressed differs greatly and that some cells (for example, AR4-2J rat pancreatoma cells) express all three receptors. Analysis of the effects of cholecystokinin and bombesin on AR4-2J cells showed that each of the InsP3 receptors could be down-regulated during activation of phosphoinositide hydrolysis, but that depletion of the type II receptor was limited. Such a discrepancy was also seen in rat cerebellar granule cells and was found to result from the type II receptor being relatively resistant to degradation. In conclusion, type I, II, and III receptors can all be down-regulated, but with different characteristics. As the relative abundance of InsP3 receptors is extremely variable, the extent to which activation of the down-regulatory process alters intracellular signaling will vary depending on which InsP3 receptors are expressed.

Calcium and a mitochondrial signal interact to stimulate phosphoinositide hydrolysis and insulin secretion in rat islets.

Fuel metabolism generates multiple signals that interact to stimulate insulin secretion. These studies explored the mechanism by which fuels activate phosphoinositide (PI) hydrolysis and the role of this signal transduction pathway in fuel-stimulated insulin secretion. High potassium (30 mM), which depolarizes the membrane and increases Ca2+ influx, caused only a transient monophasic release of insulin. In contrast, glucose (20 mM) or monomethylsuccinate (MMSucc; 10 mM) markedly stimulated a sustained insulin secretory response, indicating that fuel metabolism generates a signal(s) in addition to Ca2+ influx that is required for a sustained secretory response. On the other hand, diazoxide, an ATP-sensitive K+ channel activator that prevents membrane depolarization and Ca2+ influx in response to fuel metabolism, reduced the secretory responses to glucose and MMSucc to baseline levels, demonstrating that Ca2+ influx was essential to fuel-stimulated insulin secretion. The further addition of high K+ bypassed the diazoxide block and restored insulin secretory rates. The insulin secretory response to glucose or MMSucc in the presence of diazoxide and K+ was inhibited by the Ca2+ channel antagonist nitrendipine and the protein kinase-C inhibitor staurosporine. Changes in PI hydrolysis paralleled those in insulin secretion. High potassium alone induced only a modest 2.5-fold increase in inositol phosphate accumulation. This response was significantly less than that to glucose or MMSucc, which increased inositol phosphate accumulation by 6.8- or 5.2-fold, respectively. Like its effect on secretion, diazoxide markedly reduced glucose- or MMSucc-stimulated PI hydrolysis, and this inhibition was reversed with high K+. In contrast, diazoxide had no effect on receptor-activated PI hydrolysis stimulated by 100 nM cholecystokinin (CCK), and the effects of CCK were not dependent on added fuel, indicating that fuel and CCK activate PI hydrolysis by distinct pathways. These findings demonstrate that mitochondrial metabolism of glucose or MMSucc generates a signal(s) that interacts with Ca2+ influx to stimulate PI hydrolysis and sustained insulin secretion. This pathway of fuel-activated PI hydrolysis is distinct from that of CCK receptor-activated PI hydrolysis. These studies suggest that fuel-activated PI hydrolysis plays an important role in fuel-stimulated insulin secretion.

Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC 50 values of 2.6, 3.8 and 76 μM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca 2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca 2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Dose- and age-dependent effects of prenatal ethanol exposure on hippocampal metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis.

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal formation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP1) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP1 accumulation in 10- to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP1 accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP1 accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced receptor-dependent inositol phosphate accumulation in hypoxic myocytes.

The arrhythmogenic effects of alpha 1-adrenergic receptor agonists are enhanced in the ischemic myocardium. The present study was designed to determine whether hypoxia influences alpha 1-receptor subtype activation of phosphoinositide hydrolysis in neonatal rat ventricular myocyte cultures. Hypoxia did not alter basal inositol phosphate accumulation, but markedly increased norepinephrine-dependent inositol phosphate accumulation. This effect was apparent within 30 min and readily reversed on 30 min of reoxygenation. The response to norepinephrine reflected activation of a specific alpha 1-adrenergic receptor subtype; it was inhibited by prazosin and WB-4101 but not by chloroethylclonidine or propranolol. The density of alpha 1-adrenergic receptors identified by [125I]IBE-2254 was similar in normoxic and hypoxic myocytes. Consistent with this observation, the response to a maximal concentration of norepinephrine was enhanced by hypoxia, but the half-maximum effective dose for norepinephrine was not modified. The effects of isoproterenol to stimulate adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and of carbachol to inhibit isoproterenol-stimulated cAMP accumulation were not influenced by hypoxia. In contrast, inositol phosphate accumulation in response to carbachol or thrombin was markedly increased in hypoxic myocytes. These results demonstrate an effect of hypoxia to enhance phosphoinositide hydrolysis through a mechanism(s) distal to the receptor that may have important implications with respect to calcium overload and electrical abnormalities during myocardial ischemia.

Endothelin receptors in rat cerebellum: Activation of phosphoinositide hydrolysis is transduced by multiple G-proteins

Induction of phosphoinositide hydrolysis in rat cerebellar slices by endothelins (ET-1 and ET-3) and sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labelled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of the GTPase activity induced in this preparation indicated that phosphoinositide hydrolysis is stimulated via a functional coupling between the endothelin receptor of the ET B-R subtype and a PT-insensitive G-protein family, i.e. Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the stimulation of phosphoinositide hydrolysis by ETs and SRTXs is discussed.