Phosphoinositide Hydrolysis Response Research Articles

Contrasting Effects of Allosteric and Orthosteric Agonists on M1Muscarinic Acetylcholine Receptor Internalization and Down-regulation

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.

Serotonin 5-HT 2C receptor-mediated phosphoinositide hydrolysis in rat choroid plexus after fluoxetine and citalopram treatments

Selective serotonin reuptake inhibitors (SSRIs) bind directly to various neurotransmitter receptors. The clinical effects of SSRIs appear gradually during weeks of treatment, suggesting a role for adaptive changes in neurotransmitter receptors. Most clinically used antidepressants, e.g. fluoxetine, bind to 5-HT 2C receptors. When administered chronically, many antidepressants elicit adaptive regulation of 5-HT 2C receptors. The present study was conducted in order to determine the effects of acute and chronic fluoxetine and citalopram treatments on the density and function of 5-HT 2C receptors in the rat choroid plexus. Acute and chronic treatments followed by phosphoinositide (PI) hydrolysis assays and quantitative receptor autoradiography were performed. Acute (single-dose) treatment with neither drug significantly affected basal or 5-HT-stimulated PI hydrolysis, but acute citalopram (20 mg/kg) treatment increased both agonist and antagonist binding to 5-HT 2C receptors. Chronic (14 days) citalopram treatment (20 mg/kg) increased the maximal PI hydrolysis response by 40%, but fluoxetine lacked this effect. The present data suggest that sensitisation of 5-HT 2C receptor-mediated intracellular signal transduction may play a role in the effects of citalopram. In contrast, fluoxetine treatment does not functionally sensitise 5-HT 2C receptors. Thus, functional 5-HT 2C receptor sensitisation is not a common effect of antidepressants, but the differential effects may explain some of the pharmacodynamic differences seen with these drugs, especially upon repeated administration.

Inhibition of group I metabotropic glutamate receptor responses in vivo in rats by a new generation of carboxyphenylglycine-like amino acid antagonists

A series of novel group I metabotropic glutamate receptor (mGlu) antagonists have been designed on the basis of the 4-carboxyphenylglycine pharmacophore. The compounds are either mGlu1 receptor selective or equipotent for both mGlu1 and mGlu5 receptors and have IC 50 values ranging from 1 to 30 μM determined by phosphoinositide hydrolysis (PI) assay in vitro. All the compounds produced dose-dependent inhibition of group I mGlu receptor agonist ( RS)-3,5-dihydroxyphenylglycine (DHPG)-induced limbic seizure responses in mice with ED 50 values ranging from 9 nmol for LY393053 to 138 nmol for LY339840 after intracerebroventricular injection and were more potent than the mGlu1 receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (ED 50=477 nmol). Further antagonist actions were also demonstrated in a model of ( RS)-DHPG-induced PI hydrolysis in vivo such that LY367385 and the active cis isomer of LY393053 produced dose-dependent inhibition of PI responses in both cerebellum and hippocampus. Cis LY393053 also inhibited hippocampal PI responses when administered intraperitoneally at a dose of 30 mg/kg. These compounds define a new series of group I mGlu receptor antagonists which may serve as useful experimental tools.

Functional effects of overexpression of protein kinase C-alpha, -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3.

The rat basophilic leukemic (RBL-2H3) cell line was stably transfected with the endogenously expressed Ca2+-dependent protein kinase C-alpha (PKC-alpha) and -betaI and the Ca2+-independent delta and epsilon isoforms to study their functional roles. In addition, the Ca2+-independent PKC-eta was expressed. All transfected PKC isoforms translocated to the membrane-containing fraction in response to aggregation of the IgE-sensitized high affinity receptor for IgE (Fc epsilonRI) with the Ag dinitrophenyl(25)-BSA. All PKC transfectants, except PKC-eta, showed increased proliferative responses, and aggregation of Fc epsilonRI further enhanced the rate of proliferation. The PKC transfectants also showed increased phosphoinositide hydrolysis in response to Ag aggregation of receptors. No marked differences in the Ca2+ responses of the transfectants to Ag or thapsigargin were observed. Overexpression of PKC-alpha or -epsilon specifically inhibited receptor-dependent cytosolic phospholipase A2 (cPLA2) activity, whereas this activity was enhanced in the PKC-betaI transfectant. Analysis of the secretory response revealed that overexpression of PKC-betaI and -eta significantly enhanced secretion. A broad spectrum of cytokine mRNAs was detected in all transfectants, and overexpression of PKC-betaI significantly enhanced the receptor-dependent production of IL-2 and IL-6 mRNA. These studies identify PKC-alpha and -epsilon as negative regulators of cPLA2 activity and demonstrate the importance of PKC-beta as a positive modulator of secretion, cPLA2 activity, and cytokine production in this mast cell line.

The Thrombin Receptor Second Cytoplasmic Loop Confers Coupling to Gq-like G Proteins in Chimeric Receptors: ADDITIONAL EVIDENCE FOR A COMMON TRANSMEMBRANE SIGNALING AND G PROTEIN COUPLING MECHANISM IN G PROTEIN-COUPLED RECEPTORS

Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptor's amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled beta2-adrenergic receptor (beta2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric beta2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a beta2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and beta2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and beta2AR despite the thrombin receptor's strikingly different liganding mechanism.

The phosphotyrosine phosphatase inhibitor-phenylarsine oxide restores defective phosphoinositide hydrolysis response in anergic C3H-gld/gld lymphocytes.

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation develop both lymphadenopathy and autoimmune disease. CD4-CD8- (double negative, DN) T cells comprise the major population of T cells in mature C3H-gld/gld peripheral lymphoid tissues. These DN T cells are unresponsive to many forms of stimuli and have previously been shown to exhibit abnormally elevated levels of membrane phosphotyrosine phosphatase (PTPase) activity. In the present study, we demonstrate that IP3 production in response to mitogenic stimulation with Con A or anti-CD3 mAb (145-2CII) is significantly diminished in C3H-gld/gld lymphocytes when compared to that in congenic C3H(-)+/+ cells. The capacity to produce this second-messenger can be restored by pretreating C3H-gld/gld cells with the PTPase inhibitor, phenylarsine oxide (PAO). Although the inhibition of PTPase activity by treatment with PAO did restore C3H-gld/gld cell ability to produce IP3, the signal did not lead to lymphocyte proliferation, but instead to cell death. Our results suggest that the altered phosphoinositide hydrolysis observed in the mutant cells is related to their elevated membrane PTPase activity and that the anergy in these cells is at least in part related to the abnormally high activity of endogenous PTPases.

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.

5-HT2C receptor-mediated phosphoinositide turnover and the stimulus effects of m-chlorophenylpiperazine.

The present study was designed to investigate the hypothesis that agonist interactions at 5-HT2C receptors mediate the discriminative stimulus properties of m-chlorophenylpiperazine (mCPP). Three structural classes of compounds have been described to stimulate increases in phosphoinositide (PI) hydrolysis at the 5-HT2C receptor site: phenylpiperazines, phenylalkylamines, and indolamines. Four representative phenylpiperazines, mCPP, TFMPP, MK-212 and quipazine, one phenylalkylamine, (-)DOM, and one indolamine, LSD, were employed in the present study. The efficacies of these compounds were defined (1) in vitro, with respect to their abilities to stimulate increases in PI hydrolysis in the choroid plexus, and (2) in vivo with respect to their abilities to substitute for the mCPP discriminative stimulus. In vitro intrinsic activity at the 5-HT2C site was expressed as a fraction of the maximal PI hydrolysis response elicited by serotonin (5-HT). MK-212 (fractional efficacy = 1.1) and (-)DOM (0.77) were full agonists, while mCPP (0.72), LSD (0.27), quipazine (0.24), and TFMPP (0.22) were partial agonists with respect to the stimulation of PI hydrolysis at the 5-HT2C receptor. In vivo, each of the phenylpiperazines fully substituted for the mCPP stimulus, while (-)DOM (75%), and LSD (67%) elicited only partial substitution. While compounds with agonist activity at the 5-HT2C receptor in vitro substitute for the mCPP stimulus in vivo, no clear relationship exists between in vitro intrinsic activity at the 5-HT2C receptor with respect to the stimulation of PI turnover and maximal substitution for the mCPP stimulus in vivo. The present data suggest that mCPP elicits a compound stimulus which is mediated by agonist interactions at the 5-HT2C receptor and possibly additional interactions with 5-HT2A, 5-HT3, and/or 5-HT1B receptors.

Endothelin-induced phosphoinositide hydrolysis in the muscular layer of stem villi vessels of human term placenta

In the present study, we examined the relationship between endothelin receptors and phosphoinositide breakdown in muscle explants of placental stem villi vessels. All peptides examined, i.e. endothelin-1 (ET-1), ET-3, sarafotoxin 6b (S6b) and S6c, were able to induce phosphoinositide hydrolysis in a dose-dependent manner: ET-1 was more potent than S6b and ET-3, with corresponding EC50 values of 44 +/- 16 pmol/l, 18 +/- 13 nmol/l and 33 +/- 24 nmol/l, respectively. Sarafotoxin induced only moderate stimulation of inositol phosphate accumulation. Both ET-1- and S6b-induced accumulation of inositol phosphate was almost totally (90%) inhibited by 100 mumol/l BQ 123, while the S6c response was not affected by the ETA receptor antagonist. In contrast, the ETB receptor antagonist IRL 1038 inhibited S6c-induced inositol phosphate accumulation by more than 80%, whereas inhibition was only about 30% for ET-1 and S6b stimulations. This indicates that both ETA and ETB receptors were coupled to the phospholipase C transducing system in the muscular layer of placental stem villi vessels, and there is evidence that the phosphoinositide hydrolysis response is obtained predominantly via ETA receptor activation.

Pharmacological analysis of 4-carboxyphenylglycine derivatives: Comparison of effects on mGluR1α and mGluR5a subtypes

The antagonist effects of the 4-carboxyphenylglycines: ( S)-4-carboxy-3hydroxyphenylglycine (4C3HPG), ( S)-4-carboxyphenylglycine (4CPG) and (+)-α-methyl-4-carboxyphenylglycine (M4CPG) were compared on functional responses of human metabotropic glutamate receptor (mGluR) subtypes mGluR1α and mGluR5a. These receptors both belong to group 1 type mGluRs which couple to the phosphoinositide (PI) hydrolysis/[Ca 2+]i mobilization signal transduction pathway and are closely related in both structure and agonist pharmacology. In this study, the IC 50 values obtained for quisqualate induced PI hydrolysis responses show that although all the phenylglycines are antagonists for both mGluR1α and mGluR5a, the compounds exhibit differential potencies at these receptor subtypes. The 4C3HPG derivative was the most potent antagonist for both mGluR1α (IC 50 range: 19–50μM) and mGluR5a (IC50 range: 53–280μM). 4CPG produced an IC 50 range of 44–72μM for mGluR1α and 150–156 μM for mGluR5a cells. The potency of the M4CPG could not be distinguished from that of 4CPG with IC 50 ranges of 29–100 μM and 115–210 μM for mGluR1α and mGluR5a respectively. Further characterization of the dose-response effects of the compounds on quisqualate induced [Ca 2+]i mobilization showed that although the magnitude of phenylglycine inhibition was reduced for both mGluR subtypes compared to those observed for stimulation of PI hydrolysis (except for 4C3HPG on mGluR1α), similar differences in the relative potencies of the phenylglycines between mGluR1α (IC 50s: 40 ± 10 μM for 4C3HPG: 300–1000 μM for 4CPG and M4CPG) and mGluR5a (IC 50s: > 1000 μM) were evident. These results indicate that closely related receptors of the same mGluR group exhibit a different pharmacology with respect to phenylglycine antagonists.

Regulation of serotonin 5-HT 2C receptors in the rat choroid plexus after acute clozapine treatment

We studied the effects of acute clozapine and haloperidol treatments on 5-HT 2C receptor binding characteristics and 5-HT 2C receptor-mediated phosphoinositide hydrolysis in the rat choroid plexus. Scatchard analysis (with [ 3H]mesulergine) showed that acute clozapine treatment (10 and 25 mg/kg) decreased the density ( B max) of 5-HT 2C receptors by 20–25% with no marked change in the affinity ( K d). Quantitative autoradiography was in accordance with homogenate binding studies showing that acute clozapine treatment, unlike haloperidol (0.5 mg/kg), decreased the number of both agonist ([ 125I](±)-1-(2,5-dimethoxy-4-iodophenyl)- -2-aminopropane, [ 125I]DOI) and antagonist ([ 3H]mesulergine) labeled 5-HT 2C receptor binding sites. The decrease was more robust with the higher dose of clozapine. For comparison, both doses of clozepine, unlike haloperidol, decreased equally the density of 5-HT 2A receptors in the frontal cortex by about 45%, whereas none of the treatments altered dopamine D 2 receptor characteristics in the striatum. The K d value of 5-HT 2A receptors was significantly increased after the dose of 25 mg/kg of clozapine. These clozapine treatments failed to decrease the maximal 5-HT 2C receptor-mediated phosphoinositide hydrolysis response. The higher dose of clozapine increased 5-HT-induced phosphoinositide hydrolysis response, but also decreased significantly the basal levels of phosphoinositide hydrolysis. Haloperidol did not significantly affect the 5-HT 2C receptor-mediated phosphoinositide hydrolysis. To summarize, the present data show that a single injection of clozapine is able to reduce the density of 5-HT 2C receptors but fails to cause functional desensitization of 5-HT 2C receptors in the rat choroid plexus.

5-HT 1C receptor-mediated phosphoinositide hydrolysis in the rat choroid plexus after chronic treatment with clozapine

Chronic treatment with clozapine (14 days; 10 and 25 mg/kg/day) decreases 5-HT 1C receptor density but not affinity in rat choroid plexus measured with [ 3H]mesulergine. We now report the effects of the same clozapine treatment regimens on the function of 5-HT 1C receptors (measured by maximal stimulation of 5-HT 1C receptor-mediated phosphoinositide hydrolysis) in relation to receptor changes in rat choroid plexus. Quantitative 5-HT 1C receptor autoradiography indicated that chronic clozapine treatment decreased, in a dose-related manner, 5-HT 1C receptor binding sites labeled by antagonist ([ 3H]mesulergine) and agonist ( [ 125 I](±)-1-(2,5- dimethoxy-4- iodophenyl)-2- aminopropane, [ 125 I] DOI ) radioligands. However, only the higher dose of clozapine decreased statistically significantly the maximal 5-HT 1C receptor-mediated phosphoinositide hydrolysis response. Chronic administration of haloperidol (0.5 mg/kg/day) did not change any of the 5-HT 1C receptor parameters. In conclusion, chronic clozapine treatment is able to modulate the function of 5-HT 1C receptors. This further strengthens the possibility that 5-HT 1C receptors may contribute to some of the atypical effects of clozapine.

Alpha 1-adrenergic and arginine vasopressin stimulation of inositide hydrolysis in rat hepatocytes is unaltered in senescence.

Alpha 1-adrenergic receptors in the liver support glucose counterregulation by stimulating glycogenolysis and gluconeogenesis. We have recently reported that senescence is accompanied by reductions both in the density of alpha 1 receptors in liver homogenates and in the affinity of these receptors for agonists (Borst and Scarpace, 1990a). The present studies were undertaken to determine what consequences these reductions may have for agonist-stimulated second messenger production. To this end we measured the density of alpha 1-adrenergic receptors and agonist-stimulated phosphoinositide (PI) hydrolysis in short-term hepatocyte cultures derived from young and senescent rats. Receptor density in hepatocytes was unchanged with age. PI hydrolysis response to epinephrine was alpha 1 in nature, as it was fully blocked by prazosin. The epinephrine response was unchanged with age, both in maximum response and in sensitivity. Similarly, the response to arginine vasopressin (AVP) was also unchanged with age.

Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling

Thrombin, a protease generated at sites of vascular injury, signals cellular responses vital for hemostasis and thrombosis. How thrombin, an enzyme rather than a classical ligand, effects graded and concentration-dependent responses in its target cells has been a long-standing question. Thrombin activates its receptor by cleaving off an activation peptide to unmask a tethered peptide ligand. We utilized a thrombin receptor with an epitope-tagged activation peptide to directly demonstrate thrombin receptor cleavage and to examine the kinetics of receptor activation on intact cells. The rate of thrombin receptor cleavage was proportional to thrombin concentration over the physiologic range, but low thrombin concentrations ultimately cleaved and activated all receptors. Cumulative phosphoinositide hydrolysis in response to thrombin correlated precisely with cumulative receptor cleavage. These data strongly suggest that each cleaved and activated thrombin receptor produces a "quantum" of phosphatidylinositol hydrolysis, then shuts off. Surprisingly, this shut off occurred despite the continued presence of cleaved and "activated" receptors on the cell surface and at a time when the cells were refractory to thrombin but sensitive to agonist peptide, suggesting that a novel shut off mechanism may have evolved to deal with the tethered ligand. Unlike the case with classical ligands, cells thus cannot detect differences in thrombin concentrations as differences in fractional occupancy but rather must sense different rates of receptor activation. Because each cleaved thrombin receptor generates a quantum of second messenger, the magnitude of the cell's response to thrombin must be determined by the balance between rates of receptor activation and second messenger clearance.

The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase.

Thrombin both stimulates phosphoinositide hydrolysis and inhibits adenylyl cyclase in a variety of cell types. Whether the cloned human platelet thrombin receptor accounts for both of these signaling events is unknown. We report that thrombin receptor agonist peptide causes both phosphoinositide hydrolysis and inhibition of adenylyl cyclase in naturally thrombin-responsive CCL-39 cells. To exclude the possibility that the agonist peptide or thrombin itself may activate these pathways via distinct receptors and to circumvent a lack of suitable thrombin receptor-null cells, we utilized a designed "enterokinase receptor," a thrombin receptor with its thrombin cleavage recognition sequence LDPR replaced by DDDDK, the enterokinase cleavage recognition sequence. Transfection of enterokinase-unresponsive cells with this construct conferred both enterokinase-sensitive phosphoinositide hydrolysis and inhibition of adenylyl cyclase. The phosphoinositide hydrolysis response was largely insensitive to pertussis toxin, whereas the adenylyl cyclase response was completely blocked by pertussis toxin. These data show that the cloned thrombin receptor can effect both phosphoinositide hydrolysis and inhibition of adenylyl cyclase via at least two distinct effectors, most likely Gq-like and Gi-like G-proteins.

Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation

The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid ( trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6–11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.

Alterations of choline acetyltransferase, phosphoinositide hydrolysis, and cytoskeletal proteins in rat brain in response to colchicine administration.

Colchicine, a microtubule disrupting toxin, was administered intracerebroventricularly to rats, followed by measurements of (i) the activity of choline acetyltransferase, a biochemical marker of cholinergic neurons, (ii) cytoskeletal protein concentrations, including tau, MAP-2, spectrin, and tubulin, and (iii) the activity of the second messenger-generating system, receptor-coupled phosphoinositide hydrolysis. One week after colchicine treatment there was a 60% decrease in choline acetyltransferase activity in the hippocampus, which was followed by a gradual increase in only a 29% deficit after 12 weeks. In the striatum and cerebral cortex, choline acetyltransferase activity was slightly reduced (by 13% and 19%, respectively) 1 week after colchicine treatment followed by increases to control values. The concentrations of tau and tubulin in the hippocampus were unaltered by colchicine treatment, and MAP-2 and spectrin were only slightly reduced 4 weeks after colchicine. Hippocampal phosphoinositide hydrolysis induced by norepinephrine was elevated approximately 28% 1 and 2 weeks after colchicine treatment and that induced by ibotenate was increased by 53% 2 weeks after colchicine. These results demonstrate that colchicine causes a severe depletion of choline acetyltransferase 1 week after administration. There was not a significant reduction of the concentration of any of the cytoskeletal proteins after 1 week, possibly due to the cell-selectivity of the toxic effect of colchicine, but there was a delayed, and temporary, decline of MAP-2 and spectrin concentrations. Associated with the decreased choline acetyltransferase activity after 1 week was an enhanced phosphoinositide hydrolysis in response to norepinephrine, and after 2 weeks there were enhanced responses to norepinephrine and to ibotenate.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.

Repeated Treatment with Levoprotiline, a Novel Antidepressant, Up-Regulates Histamine Hi Receptors and Phosphoinositide Hydrolysis Response In Vivo

The effects of repeated administration of levoprotiline, a novel type of tetracyclic antidepressant on histamine H1, muscarinic acetylcholine and α1-adrenergic receptors and the response of phosphoinositide hydrolysis (PI) stimulated by histamine in the cortex of the rat brain were investigated. Histamine H1 receptors were up-regulated to 120% and PI response stimulated by histamine was enhanced to 160%–200% after repeated treatment with levoprotiline (20mg/kg, i.p., once a day for 28 days) when compared to that of the saline-treated group. No significant alterations of muscarinic acetylcholine and α1-adrenergic receptors were observed. This demonstrates that the repeated treatment with levoprotiline has prominent action on the regulation of histamine H1 receptors and PI response coupling to histamine H1 receptors in vivo.

Differential effects of propranolol on the IgE-dependent, or calcium ionophore-stimulated, phosphoinositide hydrolysis and calcium mobilization in a mast (RBL 2H3) cell line

Our previous studies demonstrated that propranolol, an inhibitor of phosphatidic acid phosphohydrolase (PAPase) (EC 3.1.3.4) blocks the IgE-dependent mediator release from a rat mast (RBL 2H3) cell line. To continue these studies, we examined the ability of propranolol to inhibit the IgE-dependent or ionomycin-mediated phosphoinositide hydrolysis and calcium mobilization in RBL 2H3 cells. RBL 2H3 cells, sensitized with mouse monoclonal anti-trinitrophenol IgE (anti-TNP IgE), were stimulated to release both histamine and peptidoleukotrienes (LT) in response to a suboptimal concentration of trinitrophenol-ovalbumin conjugate (TNP-OVA) or ionomycin. Preincubation of the cells with d, l-propranolol (300 μM) significantly (P < 0.05) inhibited the effects of both TNP-OVA and ionomycin on histamine and LT release. There was no difference in potency for the different isomers of propranolol, indicating that these effects were not a consequence of an effect on β 2-adrenergic receptors. TNP-OVA produced a rapid hydrolysis of phosphoinositides resulting in a time-dependent increase in mono- (IP 1), di- (IP 2), tri- (IP 3), and total inositol phosphate production. Ionomycin also produced a rapid increase in total inositol phosphate production; however, this largely reflected an accumulation of IP 1 Both secretagogues produced a rapid elevation in cytosolic free calcium ([Ca 2+] i); however, the effect of ionomycin maximized within a much shorter time frame than the effect of TNP-OVA. The effects of TNP-OVA on phosphoinositide hydrolysis and increase in [Ca 2+] i were inhibited by propranolol over exactly the same concentration range as the effects of this compound on TNP-OVA-stimulated mediator release. In contrast, propranolol had no effect on the increase in [Ca 2+] i and phosphoinositide hydrolysis in response to ionomycin. Taken together, these results suggest that PAPase/ phospholipase D (PLD) (EC 3.1.4.4) activation may be a prerequisite for both IgE-dependent and ionomycin-stimulated mediator release from RBL 2H3 cells. Although other explanations are possible, the data further suggest that receptor-mediated, but not ionophore-stimulated, phosphoinositide hydrolysis and [Ca 2+] i in RBL 2H3 cells may be regulated by a propranolol-sensitive pathway involving possible activation of PAPase.