Stimulation Of Phosphoinositide Hydrolysis Research Articles

Methoctramine and gallamine inhibit PI hydrolysis in guinea-pig gallbladder

The present study aimed to determine the effect of two M 2/M 4-selective muscarinic receptor antagonists on blocking the hydrolysis of carbachol (CCh) stimulated phospho-inositide (PI) breakdown in order to address the possibility that a muscarinic receptor other than the M 3 receptor is involved in PI hydrolysis in this tissue. Gallbladder tissue slices labeled with myo-[2- 3H] inositol were incubated with increasing concentrations of antagonists and agonist. After the reactions were terminated by the addition of chloroform/methanol, labeled inositol phosphates were separated using anion exchange chromatography. Muscarinic M 2 antagonists methoctramine and gallamine both inhibited carbachol-induced PI breakdown at high concentrations, with log IC 50 values of − 5.145 and − 6.049, respectively. Gallamine at 10 − 5 M concentration failed to displace the dose-response curve for carbachol-induced accumulation of inositol triphosphate (IP 3). Our data suggest that M 3 receptors play a major role in stimulation of PI hydrolysis in the guinea-pig gallbladder.

Ethological resolution of behavioural topography and D1‐like versus D2‐like agonist responses in congenic D5 dopamine receptor mutants: Identification of D5:D2‐like interactions

The phenotypic ethogram of congenic dopamine D(5) receptor "knockout" mice was evaluated. Each individual topography of behaviour within the natural repertoire was assessed over the extended course of initial exploration of and subsequent habituation to the environment, and following challenge with a series of D(1)-like agonists. Over initial exploration, D(5)-null mice evidenced a modest reduction in locomotion and a modest increase in sifting. Subsequent habituation revealed additional phenotypic effects, primarily overall reduction in grooming and delayed habituation of rearing. Among D(1)-like agonists, A 68930 stimulates both adenylyl cyclase and a putative D(1)-like receptor coupled to stimulation of phospholipase C-mediated phosphoinositide hydrolysis; conversely, SK&F 83959 stimulates phosphoinositide hydrolysis but not adenylyl cyclase while SK&F 83822 stimulates adenylyl cyclase but not phosphoinositide hydrolysis. Though programmed grooming syntax and episodic seizure activity induced by A 68930 and SK&F 83822 were unaltered, grooming induced by SK&F 83959 was reduced in D(5) mutants. Stereotyped, ponderous locomotion induced by the D(2)-like agonist RU 24213 was enhanced in D(5) mutants. Phenotypic and pharmacological characterisation of congenic D(5)-null mice at an ethological level identifies novel functional roles for the D(5) receptor in mediating discrete topographies of behaviour relating to exploration, sequential motor coordination, and how these processes change over the course of interaction with and habituation to the environment. Additionally, they indicate the involvement of phosphoinositide hydrolysis and D(5):D(2)-like interactions in regulating these processes.

Interleukin-1β down-regulates the expression of metabotropic glutamate receptor 5 in cultured human astrocytes

Expression of metabotropic glutamate receptor 5 (mGluR5) protein is known to be plastic and to depend critically on the astrocytes' microenvironment. In the present study we investigated whether interleukins, which are involved in the immune response following brain injury, could contribute to the regulation of mGluR5 protein in human astrocytes in culture. Using Western blotting and immunocytochemistry, no detectable changes in the expression of the mGluR5 protein were observed with both interleukin 1β and interleukin 6 in undifferentiated cultures (growing in serum free media). In contrast, in cultures that had been morphologically differentiated by exposure to epidermal growth factor (EGF), addition of interleukin 1β (but not interleukin 6) reduced mGluR5 protein expression. In addition, stimulation of phosphoinositide hydrolysis by the selective group I agonist ( S)-3,5-dihydroxyphenylglycine (DHPG) was reduced after exposure to interleukin 1β. The suppressive effect on mGluR5 was prevented by the interleukin 1 receptor antagonist. Thus, interleukin 1β may represent an additional pathway through which mGluR5 expression and function can be modulated in astrocytes under different pathological conditions associated with an inflammatory response.

High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain

Ontogenetic studies indicate that inositol phosphate accumulation in rodent brain tissue by cholinergic muscarinic agonists as well as expression of high-affinity neurotensin receptor (NTS1) peak at 7 days after birth. Herein, potential participation of this receptor in such effect was investigated. Cerebral cortex prisms of 7-day-old rats were preloaded with [ 3H] myoinositol and later incubated during 60 or 20 min in the presence of muscarinic agonist carbachol plus neurotensin and SR 48692, a non-peptide NTS1 antagonist. In 60-min incubation experiments, inositol phosphate accumulation by 10 −3 M carbachol was roughly 320%, an effect which remained unaltered plus 10 −6 M to 10 −4 M neurotensin but partially decreased with equimolar SR 48692 concentration. In 20-min incubation experiments, inositol phosphate accumulation by 10 −3 M carbachol was circa 240%, a value which attained 320–360% plus 10 −7 M neurotensin; this effect was totally blocked by 10 −7 M SR 48692. It was concluded that in inositol phosphate accumulation by carbachol, besides the cholinergic muscarinic receptor, the NTS1 receptor is likewise involved; findings at 60 min are attributable to the effect of endogenous neurotensin whereas those at 20 min most likely involve both endogenous and exogenously added peptide.

Activation of PLC-δ1 by Gi/o-coupled receptor agonists

The mechanism of phospholipase (PLC)-delta activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca(2+) stimulated an eightfold increase in PLC-delta1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G(i/o)-coupled receptor agonists (somatostatin, delta-opioid agonist [D-Pen(2),D-Pen(5)]enkephalin, and A(1) agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-delta1(E341R/D343R; 65-76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca(2+) influx and was not observed in the absence of extracellular Ca(2+), but was partly inhibited by nifedipine (16-30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca(2+) channels. Treatment of the cells with a G(q/13)-coupled receptor agonist, CCK-8, caused only transient, PLC-beta1-mediated PI hydrolysis. Unlike G(i/o)-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-delta1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Galpha(13) minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-delta1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca(2+) influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine ( approximately 25%) and strongly inhibited by SKF-96365 ( approximately 75%) and in cells expressing PLC-delta1(E341R/D343R). Agonist-independent Ca(2+) release or Ca(2+) influx via voltage-gated Ca(2+) channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-delta1 antibody or nifedipine. We conclude that PLC-delta1 is activated by G(i/o)-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca(2+) influx via store-operated Ca(2+) channels.

Prenatal Ethanol Exposure Reduces mGluR5 Receptor Number and Function in the Dentate Gyrus of Adult Offspring

Previous studies in our laboratory indicated that metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis is markedly reduced in the hippocampal formation of adult rat offspring whose mothers drank moderate amounts of ethanol during pregnancy. In the present study, we extended these observations by measuring the impact of prenatal ethanol exposure on proteins associated with the mGluR5 receptor-effector system along with two mGluR5 agonist-mediated responses in dentate gyrus of adult offspring. Sprague-Dawley rat dams consumed one of three diets throughout gestation: (1) a BioServ liquid diet that contained 5% ethanol (v/v), (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet, or (3) lab chow ad libitum. Microdissected slices of dentate gyrus were prepared from adult female offspring from each diet group and used for (1) Western blot analyses of mGluR5, the G-proteins Galphaq and Galpha11, and phospholipase C-beta1; (2) 2-chloro-5-hydroxyphenylglycine (CHPG)-stimulated growth associated protein 43 (GAP-43) phosphorylation; or (3) CHPG potentiation of electrically evoked [H]-D-aspartate (D-ASP) release from dentate gyrus slices. In tissue prepared from untreated control rats, CHPG produced a dose-dependent increase in phosphate incorporation into GAP-43, with maximal agonist stimulation occurring at 20 microM of CHPG. CHPG produced a quantitatively similar dose-dependent increase in the potentiation of electrically evoked D-ASP release from dentate gyrus slices from untreated controls. Fetal ethanol exposure reduced the amount of dentate gyrus mGluR5 receptor protein by 36% compared with the diet control groups. There were no significant differences between diet groups in the two G-proteins or phospholipase C-beta1 protein. Fetal ethanol exposure reduced CHPG-stimulated GAP-43 phosphorylation to approximately one half the amount of CHPG stimulation observed in the control diet groups. Prenatal ethanol exposure also reduced CHPG potentiation of D-ASP release to a similar degree compared with control. These results indicate that prenatal exposure to moderate quantities of ethanol reduces mGluR5 expression in the dentate gyrus of adult offspring. Although the subcellular site(s) for reduced mGluR5 expression cannot be discerned from Western blot data, the quantitatively similar effects of prenatal ethanol exposure on mGluR5 agonist stimulation of presynaptically localized GAP-43 phosphorylation and CHPG potentiation of evoked D-ASP release suggest that the presynaptic nerve terminal is one site where prenatal ethanol exposure has reduced mGluR5 receptor number and function. Furthermore, these data implicate these neurochemical alterations as one factor contributing to the hippocampal synaptic plasticity and behavioral deficits that we have observed previously in prenatal ethanol-exposed offspring.

Autoantibodies against cerebral muscarinic cholinoceptors in Sjögren syndrome: functional and pathological implications

Previous studies have demonstrated that antibodies against muscarinic acetylcholine receptors (mAChRs) from exocrine glands, correlates with Sjögren syndrome (SS) in the majority of patients. The aim of the present investigation was to establish if serum IgG antibodies present in SS interacts with cerebral mAChRs. Results show that anti-cerebral IgG are present in the sera of 40% SS patients studied. Autoantibodies were able to interact with mAChRs of cerebral frontal cortex membranes inhibiting the [ 3H]QNB binding to its specific receptor. Moreover, tested by ELISA and dot blot they recognized the synthetic peptides corresponding to the second extracellular loop of human M 1 and M 3 mAChR. In addition, the corresponding affinity-purified anti-M 1 and anti-M 3 peptide IgGs displayed an agonistic activity, stimulating phosphoinositide hydrolysis. The results support the notion that serum IgG autoantibodies in SS patients target cerebral mAChRs may have some role in the pathogenesis of higher cognitive dysfunction present in SS patients.

L-homocysteine sulfinic acid and L-homocysteic acid stimulate phosphoinositide hydrolysis in rat cortical neurons.

L-homocysteine sulfinic acid and L-homocysteic acid stimulate phosphoinositide hydrolysis in rat cortical neurons.

Pharmacological Analysis of the Contractile Role of M 2 and M 3 Muscarinic Receptors in Smooth Muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M 2 and M 3 subtypes. The M 3 subtype triggers contraction through an interaction with G q proteins to stimulate phosphoinositide hydrolysis and mobilize Ca 2+ . In contrast, activation of M 2 receptors modulates contraction by preventing relaxation or by potentiating M 3 receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M 2 receptors to G i proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M 2 and M 3 receptors has been shown to resemble the profile of the directly acting receptor (M 3 ), primarily, and not that of the conditional receptor (M 2 ). Evidence for a contractile role of the M 2 receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M 3 receptors have been covalently inactivated. Under these conditions, M 2 receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M 2 and M 3 receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.

Metabotropic glutamate receptor 5 (mGluR5)-mediated phosphoinositide hydrolysis and NMDA-potentiating effects are blunted in the striatum of aged rats: a possible additional mechanism in striatal senescence.

The aim of the present work was to verify whether an impairment of subtype 5 metabotropic glutamate receptor-mediated neurotransmission did occur in the aged striatum. To this end, the ability of the subtype 5 metabotropic glutamate receptor agonist, RS-2-chloro-5-hydroxyphenylglycine, to stimulate phosphoinositide hydrolysis and to potentiate N-methyl-d-aspartate-induced effects in striatal slices from young (3 months) and aged (24 months) rats was compared. The ability of RS-2-chloro-5-hydroxyphenylglycine to induce maximal phosphoinositide turnover and to potentiate N-methyl-d-aspartate effects was significantly reduced in slices from old vs. young rats. These changes were associated with a significant reduction in the expression of subtype 5 metabotropic glutamate receptor protein (-28.8%) and phospholipase C-beta1 (-55.8%) in old striata, while receptor messenger ribonucleic acid expression was unchanged. These results show that the signalling associated with subtype 5 metabotropic glutamate receptors undergoes significant age-related changes and that a reduced expression of subtype 5 metabotropic glutamate receptors and, more importantly, phospholipase C-beta1 may account for the functional decline of subtype 5 metabotropic glutamate receptors.

Comparison of the pharmacological antagonism of M 2 and M 3 muscarinic receptors expressed in isolation and in combination

We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M 2 and M 3 muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [ 3 H ] N-methylscopolamine binding to CHO cells expressing both M 2 and M 3 muscarinic receptors (CHO M 2+M 3 cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M 2 cells, whereas the other site exhibited properties like those of CHO M 3 cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M 3 and CHO M 2+M 3 cells, but not in CHO M 2 cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M 3 and CHO M 2+M 3 cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M 2 and CHO M 2+M 3 cells and a less robust inhibition in CHO M 3 cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M 3 and CHO M 2+M 3 cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M 2, CHO M 3, and CHO M 2+M 3 cells. The pharmacological antagonism of the cAMP response in CHO M 2+M 3 cells resembled that expected for a response mediated independently by both M 2 and M 3 receptors.

L-homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists.

Moderate hyperhomocysteinemia is associated with several diseases, including coronary artery disease, stroke, Alzheimer's disease, schizophrenia, and spina bifida. However, the mechanisms for their pathogenesis are unknown but could involve the interaction of homocysteine or its metabolites with molecular targets such as neurotransmitter receptors, channels, or transporters. We discovered that L-homocysteine sulfinic acid (L-HCSA), L-homocysteic acid, L-cysteine sulfinic acid, and L-cysteic acid are potent and effective agonists at several rat metabotropic glutamate receptors (mGluRs). These acidic homocysteine derivatives 1) stimulated phosphoinositide hydrolysis in the cells stably expressing the mGluR1, mGluR5, or mGluR8 (plus Galpha(qi9)) and 2) inhibited the forskolin-induced cAMP accumulation in the cells stably expressing mGluR2, mGluR4, or mGluR6, with different potencies and efficacies depending on receptor subtypes. Of the four compounds, L-HCSA is the most potent agonist at mGluR1, mGluR2, mGluR4, mGluR5, mGluR6, and mGluR8. The effects of the four agonists were selective for mGluRs because activity was not discovered when L-HCSA and several other homocysteine derivatives were screened against a large panel of cloned neurotransmitter receptors, channels, and transporters. These findings imply that mGluRs are candidate G-protein-coupled receptors for mediating the intracellular signaling events induced by acidic homocysteine derivatives. The relevance of these findings for the role of mGluRs in the pathogenesis of homocysteine-mediated phenomena is discussed.

Pharmacological Analysis of the Contractile Role of M 2 and M 3 Muscarinic Receptors in Smooth Muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M(2) and M(3) subtypes. The M(3) subtype triggers contraction through an interaction with G(q) proteins to stimulate phosphoinositide hydrolysis and mobilize Ca(2+). In contrast, activation of M(2) receptors modulates contraction by preventing relaxation or by potentiating M(3) receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M(2) receptors to G(i) proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M(2) and M(3) receptors has been shown to resemble the profile of the directly acting receptor (M(3)), primarily, and not that of the conditional receptor (M(2)). Evidence for a contractile role of the M(2) receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M(3) receptors have been covalently inactivated. Under these conditions, M(2) receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M(2) and M(3) receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.

Chronic methamphetamine administration reduces histamine-stimulated phosphoinositide hydrolysis in mouse frontal cortex

In the present study, it was hypothesized that in vivo pretreatment with repeated methamphetamine would alter the agonist-stimulated phosphoinositide hydrolysis in mouse frontal cortical slices. Male ICR mice that received the methamphetamine injection (1.0 mg/kg, intraperitoneally) once a day for five consecutive days showed behavioral sensitization to the same dose of methamphetamine 5 days after the last injection of the initial chronic treatment regimen (test day 10). On test day 10, the reduction of histamine (0.1–1.0 mM)-stimulated phosphoinositide hydrolysis in the mouse frontal cortex was observed. The reduction was specific to histamine, but not to norepinephrine ( 10 μM –0.1 mM) or l-glutamate (0.1–0.5 mM). The reduction occurred without any change in the expression level of histamine H 1 receptor mRNA. The reduction recovered 25 days after the last injection of the initial chronic treatment regimen (test day 30). The direct application to the slices of a pharmacologically effective concentration of methamphetamine in vitro (10 μM) did not alter the histamine signal transduction. The present results suggest that the reduction is probably one of neuroadaptations in the frontal cortex contributing to behavioral sensitization.

Metabotropic glutamate receptor involvement in phosphoinositide hydrolysis stimulation by an endogenous Na +, K +-ATPase inhibitor and ouabain in neonatal rat brain

The mechanism of action of an endogenous Na +, K +-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in neonatal rat brain cortex and compared with that of ouabain. Lack of additivity for endobain E and glutamate paired stimulation on inositol phosphates accumulation suggested that they share at least a common step on inositol phosphate metabolism, as previously advanced for ouabain. In addition, Cd 2+ sensitivity of endobain E and ouabain effects strengthened the involvement of glutamate receptors. The participation of ionotropic glutamate receptors on endobain E- and ouabain-induced phosphoinositide hydrolysis seems untenable, since antagonists dizocilpine and CNQX proved unable to inhibit these effects. However, the endobain E effect was blocked by 2×10 −4 M L-AP3 (an antagonist for group I mGluRs) when at least a 15-min preincubation protocol was employed. Maximal inhibition of endobain E effect (42%) occurred when L-AP3 preincubation was extended to 60 min, as already shown with glutamate, but only a trend to decrease was recorded with ouabain. At variance, the ouabain effect was reduced to 50% employing 5×10 −4 M MCPG (a competitive antagonist for group I mGluRs), whereas no blockade was observed with endobain E or glutamate. In addition, MPEP (a selective mGluR5 antagonist) partially reduced ouabain, endobain E and glutamate responses and the selective mGluR1 antagonist LY367385 showed no activity at all. To sum up, the present findings support the involvement of mGluR5 in both endobain E and ouabain phosphoinositide hydrolysis stimulation in neonatal rat brain, in spite of dissimilar response to tested antagonists.

Protease-activated receptor-1-mediated DNA synthesis in cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in cardiac fibroblasts and cardiomyocytes.

Proteases elaborated by inflammatory cells in the heart would be expected to drive cardiac fibroblasts to proliferate, but protease-activated receptor (PAR) function in cardiac fibroblasts has never been considered. This study demonstrates that PAR-1 is the only known PAR family member functionally expressed by cardiac fibroblasts and that PAR-1 activation by thrombin leads to increased DNA synthesis in cardiac fibroblasts. The increase in DNA synthesis induced by PAR-1 substantially exceeds the effects of other G protein-coupled receptor agonists in this cell type. PAR-1 stimulates phosphoinositide hydrolysis and mobilizes intracellular calcium via pertussis toxin (PTX)-sensitive and PTX-insensitive pathways. Activation of PAR-1 leads to an increase in Src, Fyn, and epidermal growth factor receptor (EGFR) phosphorylation, with EGFR receptor transactivation by Src family kinases the major mechanism for PAR-1-dependent activation of extracellular signal-regulated kinase, p38-mitogen-activated protein kinase, and protein kinase B. Activation of PAR-1 also leads to an increase in DNA synthesis. PAR-1 signaling is highly contextual in nature, inasmuch as PAR-1 activates extracellular signal-regulated kinase and only weakly stimulates protein kinase B via a pathway that does not involve EGFR transactivation in cardiomyocytes. PAR-1 responses in cardiac fibroblasts and cardiomyocytes are predicted to contribute importantly to remodeling during cardiac injury and/or inflammation.

5-HT 2A receptor-stimulated phosphoinositide hydrolysis in the stimulus effects of hallucinogens

The role of 5-HT 2A-mediated stimulation of phosphoinositide hydrolysis in the discriminative effects of hallucinogens was investigated in PC12 cells stably expressing the rat 5-HT 2A receptor (PC12-5-HT 2A cells). The hallucinogenic compounds, d-lysergic acid diethylamide (LSD), (−)2,5-dimethoxy-4-methylamphetamine (DOM), psilocybin, N, N-dimethyltryptamine (DMT), 5-methoxy- N, N-dimethyltryptamine (MDMT) and N, N-diethyltryptamine (DET), all caused a concentration-dependent increase in the generation of [ 3H]inositol phosphates. The nonhallucinogenic compounds, 6-fluoro- N, N-diethyltryptamine (6-F-DET), lisuride and quipazine, also displayed significant efficacy in stimulating phosphoinositide hydrolysis, while 2-bromo-lysergic acid diethylamide (BOL), which is not a hallucinogen, did not alter inositol phosphate generation. The β-carbolines, harmaline and harmane, also did not alter phosphoinositide hydrolysis. Comparison of these results with previous drug discrimination studies indicated the apparent lack of correlation between the degree of substitution in LSD- and DOM-trained animals and efficacy in stimulating phosphoinositide hydrolysis. The present study indicates that 5-HT 2A-mediated stimulation of phosphoinositide hydrolysis does not appear to be the sole critical signaling mechanism involved in the discriminative effects of hallucinogens.

Stimulus generalization by fenfluramine in a quipazine–ketanserin drug discrimination is not dependent on indirect serotonin release

The purpose of this study was to determine if animals trained to discriminate a serotonin 2A (5-HT 2A) receptor agonist from a 5-HT 2A receptor antagonist would also be sensitive to alterations in serotonin neurotransmission brought about by 5-HT reuptake inhibitors and releasers. Previous work from our laboratory has shown that the quipazine–ketanserin discrimination is mediated solely by the 5-HT 2A receptor, thus providing a behavioral continuum of 5-HT 2A receptor function. Rats were trained to discriminate quipazine (0.35 mg/kg) from ketanserin (1.0 mg/kg) on a variable interval-30 schedule of reinforcement. Following acquisition, substitution tests were conducted with the training drug, quipazine, and agents that have been shown to alter the synaptic levels of 5-HT, including fenfluramine, norfenfluramine, 5-methoxy-6-methyl-2-aminoindan (MMAI) and fluoxetine. All compounds substituted, except fluoxetine. Antagonist tests with mianserin and MDL 100,907 indicated that fenfluramine's and MMAI's substitution for quipazine was mediated by the 5-HT 2A receptor. Animals were pretreated with PCPA to determine whether 5-HT release or direct agonism mediated the discriminative stimulus effects of fenfluramine and MMAI. PCPA blocked the substitution of MMAI but not of fenfluramine for quipazine. Analysis of 3H-IP formation in cells showed that norfenfluramine dose-dependently stimulated phosphoinositide hydrolysis to levels similar to that of serotonin and quipazine. These results indicate that fenfluramine's substitution for quipazine in rats trained on a quipazine–ketanserin discrimination are due to direct agonism at the 5-HT 2A receptor likely mediated by norfenfluramine, an active metabolite.

Regulatory properties of alpha(1B)-adrenergic receptors defective in coupling to phosphoinositide hydrolysis.

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for alpha(1B)-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The Delta12 and Delta5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (Delta[246-261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by alpha(1B)-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.

Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells.

The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca(2+) mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.