Phosphoinositol Hydrolysis Research Articles

Label-Free Dynamic Mass Redistribution Reveals Low-Density, Prosurvival α1B-Adrenergic Receptors in Human SW480 Colon Carcinoma Cells.

Small molecules that target the adrenergic family of G protein-coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional α1B-adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous α1B-ARs are not detectable via either [3H]-prazosin-binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of α1B-ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating β-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both α1B- and β-ARs may represent valuable small-molecule therapeutics for the treatment of colon cancer.

Nitric oxide synthase inhibition reverts muscarinic receptor down-regulation induced by pilocarpine- and kainic acid-evoked seizures in rat fronto-parietal cortex

We investigated how nitric oxide (NO) synthase inhibitor modulates muscarinic receptor expression in epileptic rats. We found that subchronic treatment (4 days) with Nω-nitro-l-arginine reduced the down-regulation of muscarinic receptors induced by pilocarpine and kainic acid in rat fronto-parietal cortex, notwithstanding the dramatic potentiation of seizures induced by both convulsants. Furthermore, functional experiments in fronto-parietal cortex slices, showed that Nω-nitro-l-arginine reduces the down-regulating effect of pilocarpine on carbachol-induced phosphoinositol hydrolysis. Finally, Nω-nitro-l-arginine greatly potentiated the induction of basic fibroblast growth factor (FGF2) by pilocarpine. These data suggest a potential role of NO in a regulatory feedback loop to control muscarinic receptor signal during seizures. The dramatic potentiation of convulsions by NO synthase inhibitors in some animal models of seizures could derive from preventing this feedback loop.

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Synthesis and Simple 18F-Labeling of 3-Fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile as a High Affinity Radioligand for Imaging Monkey Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography

2-fluoromethyl analogs of (3-[(2-methyl-1,3-thiazol-4yl)ethynyl]pyridine) were synthesized as potential ligands for metabotropic glutamate subtype-5 receptors (mGluR5s). One of these, namely, 3-fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile (3), was found to have exceptionally high affinity (IC50 = 36 pM) and potency in a phosphoinositol hydrolysis assay (IC50 = 0.714 pM) for mGluR5. Compound 3 was labeled with fluorine-18 (t1/2 = 109.7 min) in high radiochemical yield (87%) by treatment of its synthesized bromomethyl analog (17) with [18F]fluoride ion and its radioligand behavior was assessed with positron emission tomography (PET). Following intravenous injection of [18F]3 into rhesus monkey, radioactivity was avidly taken up into brain with high uptake in mGluR5 receptor-rich regions such as striata. [18F]3 was stable in monkey plasma and human whole blood in vitro and in monkey and human brain homogenates. In monkey in vivo, a single polar radiometabolite of [18F]3 appeared rapidly in plasma. [18F]3 merits further evaluation as a PET radioligand for mGluR5 in human subjects.

Role of the peptide linker in metabotropic glutamate receptor‐1 activation process

Family 3 G-protein-coupled receptors (GPCRs) consist of the extracellular calcium-sensing receptor (CaR), metabotropic glutamate receptors (mGluR1-8), γ-aminobutyric acid receptor (GABABR), taste receptors (TIR1-3), pheromone receptors (V2Rs) and orphan receptors. These are characterized by a large amino-terminal extracellular ligand binding domain (ECD) that is responsible for signaling and activation involving the seven-transmembrane helical domains (7TMD). The solved crystal structure for the closed conformation of mGluR1 indicates the carboxy-terminal portions of the monomeric units are drawn together but the signaling mechanism remains unsolved. A specific 14-amino acid linker segment of the mGluR1 connecting the ECD to the 7TMD, present in all Family 3 GPCRs except GABABR, was critical for signaling and is consistent with the peptide-linker model of receptor activation. To determine the relevance of this conserved motif in other Family 3 GPCRs, we altered the length and composition of the mGluR1 linker via insertion, deletion, and site-directed mutagenesis. Phosphoinositol hydrolysis assays, ligand binding assays and calcium imaging analyses of chimeric receptors suggest that the mGluR1 linker is critical for signaling from the ECD to the 7TMD and is consistent with our finding for the CaR, supporting the peptide-linker activation model for receptors containing this conserved motif. Work supported by the Intramural Research program of the NIDCD, National Institutes of Health.

Regulation of 5-HT 2A/C receptors and DOI-induced behaviors by protein kinase Cγ

Protein kinase Cγ (PKCγ) null mutant mice demonstrate increased behavioral impulsivity and ethanol consumption. Pharmacological studies have shown that 5-HT 2A/C receptors modulate impulsivity and ethanol consumption in rodents and that PKC can regulate 5-HT 2A/C receptors. To determine whether PKCγ plays a selective role in 5-HT 2A/C receptor regulation, biochemical and behavioral experiments were performed in PKCγ mutant and wild-type mice. DOI-stimulated phosphoinositol hydrolysis and [ 125I]-DOI saturation binding in the PFC, and quantitative autoradiography of [ 125I]-DOI binding sites in 15 brain regions were analyzed. DOI-induced head twitch responses (HTR) were measured in naive mice after an acute 2.5 mg/kg injection of DOI. Results indicated that DOI-induced HTR was significantly greater in mutant mice compared to wild-type mice. Results of the phosphoinositol hydrolysis, membrane binding, and autoradiography experiments indicated that in mutant mice, increased HTR was associated with increased 5-HT 2A/C receptor function in the PFC, but not increased receptor number or affinity suggesting that PKCγ regulates receptor function but not receptor number. These data support a role for 5-HT 2A/C receptors in the PFC in mediating some of the behavioral differences observed between PKCγ mutant and wild-type mice.

New Allosteric Modulators of Metabotropic Glutamate Receptor 5 (mGluR5) Found by Ligand‐Based Virtual Screening

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Its long-term modulating affect on synaptic transmission is mediated by the metabotropic glutamate receptors (mGluR), a family of class III G protein-coupled receptor (GPCR) proteins. This class of GPCRs is characterized by the typical seven transmembrane domain and an additional large extracellular ligand-binding domain. Allosteric modulators that bind to the receptor within the seven transmembrane helix region—that is, the region where the ligand-binding pocket is located in type I and type II GPCRs— represent an attractive class of molecules for addressing several neural disorders associated with mGluR activity. The first selective allosteric antagonists for mGluR5, SIB-1751 (1) and SIB-1893 (2), were published in 1999 (Scheme 1). SIB-1751 was identified by high-throughput screening (HTS), and SIB1893 resulted from a UNITY search for analogues. In phosphoinositol (PI) hydrolysis assays, the two molecules revealed IC50 values of 3.1 and 2.3 mm, respectively. Chemical variation of SIB-1893 resulted in the much more potent, highly selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP, 3), which had an IC50 of 36 nm in PI hydrolysis assays. [4] Several MPEP analogues (4–9) with reported low nanomolar activity have been published in the scientific and patent literature since then. Nonetheless, the mode of action of these ligands is not completely understood. Recent publications of MPEP and MPEP derivatives also reported off-target activity and a short plasma half life. In particular, the latter could be attributed to potential metabolic instability of the ethynyl linker. Pharmacophore-based similarity searching has been proven to be suited to finding new ligands that exhibit similar biological activity but are based on a different chemical scaffold. Using a set of known specific allosteric antagonists of mGluR5 (3–9), 5] which were compiled from scientific and patent literature, we applied a hierarchical, ligand-based virtual-screening approach to identify novel compounds that modulate mGluR5. First, a “drug-likeness” estimation by an artificial neural-network system was employed to prescreen just for molecules with a predicted “drug-like” structure. For subsequent similarity searching, we used the CATS3D descriptor, a 3D extension of the original topology-based CATS approach, both of which were designed for the sake of “scaffold-hopping”. 10] CATS3D encodes the conformation of a molecule in the form of a histogram that contains the normalized frequencies of all pairs of atoms of a molecule. Atom pairs were subdivided into groups that were characterized by atom–atom distance ranges in Euclidean space and six different pharmacophore types. 20 equal-distance bins from 0–20 were used. One of the pharmacophore types—cation, anion, hydrogenbond acceptor, hydrogen-bond donor, polar (hydrogen-bond acceptor and hydrogen-bond donor) or hydrophobic—was assigned to each atom by using the ph4_aType function of MOE (Chemical Computing Group Inc. , Montr al, Canada; URL: http://www.chemcomp.com). The CATS3D representation is rotationand translation-invariant, and therefore enables rapid pair-wise comparisons of molecules without the need to explicitly align the molecules. To form a hypothesis about receptor-bound 3D conformations of 3–9, we used the flexible-alignment tool of MOE. Ligands were successively aligned from 3 to 9 (Figure 1), and conformations were chosen based on existing knowledge

Endothelium-derived nitric oxide modulates vascular action of aldosterone in renal arteriole.

We have recently demonstrated that aldosterone causes nongenomic vasoconstriction by activating phospholipase C (PLC) in the preglomerular afferent arteriole (Af-Art). In the present study, we tested the hypothesis that endothelium modulates this vasoconstrictor action by releasing nitric oxide (NO). In addition, to study the post-PLC mechanism, we examined possible contributions of phosphoinositol hydrolysis products. Rabbit Af-Arts were microperfused at 60 mm Hg in vitro, and increasing doses of aldosterone (10(-10) to 10(-8) mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (P<0.01) constriction was observed from 5x10(-9) mol/L, and at 10(-8) mol/L, intraluminal diameter decreased by 29%+/-3% (n=9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10(-10) mol/L, and at 10(-8) mol/L, the diameter decreased by 38%+/-2% (n=6). NO synthesis inhibition reproduced this augmentation (n=7). Pretreatment with chelerythrine (10(-6) mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10(-8) mol/L now decreased the diameter by 18%+/-3% (n=7). However, in Af-Arts treated with thapsigargin (10(-6) mol/L) or dantrolene (3x10(-5) mol/L), which blocks inositol 1,4,5-triphosphate (IP3)-induced intracellular calcium release, aldosterone at 10(-8) mol/L decreased the diameter by only 9%+/-1% (n=6) or 9%+/-2% (n=5), respectively. These results demonstrate that in the Af-Art endothelium-derived NO modulates vasoconstrictor actions of aldosterone that are mediated by the activation of both IP3 and PKC pathways. Such vasoconstrictor actions of aldosterone may contribute to the development or aggravation of hypertension by elevating renal vascular resistance in cardiovascular diseases associated with endothelium dysfunction.

Age-dependent effects of Aroclor 1254 R on calcium uptake by subcellular organelles in selected brain regions of rats

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca 2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca 2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254 R, on 45Ca 2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90–120 (adult) male and female Long–Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca 2+-uptake in selected brain regions increased with age; PND 7<PND 21≤adults. Among three brain regions, hippocampus had relatively lower microsomal 45Ca 2+-uptake than cerebellum and frontal cortex throughout the development. Mitochondrial 45Ca 2+-uptake was comparable in three brain regions of PND 7 and adult animals, but in PND 21 rats, the cerebellum had much higher activity than frontal cortex and hippocampus. No gender-related differences were seen in 45Ca 2+-uptake by either microsomes or mitochondria in selected brain regions throughout development. Inhibition of 45Ca 2+-uptake by Aroclor 1254 in a concentration-dependent manner was observed throughout the study. However, the degree of inhibition of microsomal 45Ca 2+-uptake in these brain regions by Aroclor 1254 increased with age, PND 7<PND 21≤adults (IC 50s=21–34, 8–20 and 10–14 μM, respectively). Brain region-specific differential sensitivity to Aroclor 1254 on the inhibition of microsomal 45Ca 2+-uptake was not seen in PND 7 and adult animals but in PND 21 rats, hippocampus was more sensitive than the other selected brain regions. There were no age-, gender- or brain region-specific differential effects of Aroclor 1254 on mitochondrial 45Ca 2+-uptake. These results indicate that a commercial PCB mixture, Aroclor 1254, inhibited 45Ca 2+-uptake by both microsomes and mitochondria uniformly in selected brain regions of males and females during development. However, the inhibition of microsomal 45Ca 2+-uptake by Aroclor 1254 increased with age. The age- and gender-related differential sensitivity to Aroclor 1254 may be attributed to the changes in calcium homeostasis in various brain regions during development.

Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism.

1. The metabotropic glutamate receptors (mGluRs) are a family of G-protein linked receptors that can be divided into three groups (group I, II and III). A number of studies have implicated group I mGluR activation in acute neuronal injury, but until recently it was not possible to pharmacologically differentiate the roles of the two individual subunits (mGluR1 and mGluR5) in this group. 2. We investigated the role of mGluR5 in acute NMDA and glutamate mediated neurodegeneration in cultured rat cortical cells using the mGluR5 antagonists MPEP and SIB-1893, and found that they provide significant protection at concentrations of 20 or 200 microM. 3. These compounds act as effective mGluR5 antagonists in our cell culture system, as indicated by the ability of SIB-1893 to prevent phosphoinositol hydrolysis induced by the specific mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG). 4. However, they also significantly reduce NMDA evoked current recorded from whole cells voltage clamped at -60 mV, and significantly decrease the duration of opening of NMDA channels recorded in the outside out patch configuration. 5. This suggests that although MPEP and SIB-1893 are effective mGluR5 antagonists, they also act as noncompetitive NMDA receptor antagonists. Therefore, the neuroprotective effects of these compounds are most likely mediated through their NMDA receptor antagonist action, and caution should be exercised when drawing conclusions about the roles of mGluR5 based on their use.

Effect of phospholipase C and protein kinase C following cholinergic denervation and hippocampal sympathetic ingrowth in rat hippocampus

Following cholinergic denervation of the hippocampus by medial septal lesions, an unusual neuronal reorganization occurs in which peripheral adrenergic fibers arising from the superior cervical ganglia grow into the hippocampus (hippocampal sympathetic ingrowth). We have reported previously that cholinergic denervation and hippocampal sympathetic ingrowth differentially affected cholinergically stimulated phosphoinositide hydrolysis, concentration and affinity of muscarinic receptors, Go-protein level and protein kinase C activity. To complete these studies, we determined whether cholinergic denervation and hippocampal sympathetic ingrowth influenced phospholipase C and protein kinase C expression in dorsal hippocampal membranes and cytosol. Using immunoblotting methods, the results showed that the 100,000 mol. wt subunit of phospholipase Cβ was increased in the membrane fraction in the hippocampal sympathetic ingrowth group by 45% compared to controls and the 150,000 mol.wt subunit was increased by 75% and 59% compared to controls and cholinergic denervation, respectively. For protein kinase C detection, immunoblots were prepared using antibodies selective for “classical” protein kinase C members (α, β, γ) and for the “novel” protein kinase C subfamily members (δ, θ). Membrane protein kinase Cβ was decreased in hippocampal sympathetic ingrowth by 35% compared to controls and by 41% compared to cytosolic hippocampal sympathetic ingrowth. Membrane protein kinase Cβ was decreased in cholinergic denervation by 28% compared to controls. When compared to membranes from controls and the cholinergic denervation group, and to cytosolic fractions from the hippocampal sympathetic ingrowth groups, respectively, the following membrane protein kinase isoforms were found to be decreased by hippocampal sympathetic ingrowth: γ by 55%, 40% and 57%; δ by 91.5%, 70% and 120%; θ by 95%, 100% and 86%. In conclusion, our results may indicate the connection between the previously reported differential influence of hippocampal sympathetic ingrowth and cholinergic denervation on cholinergically stimulated phosphoinositol hydrolysis. The “normalization” of phosphoinositol hydrolysis found in hippocampal sympathetic ingrowth may be due to the increase in phospholipase Cβ expression in hippocampal sympathetic ingrowth membrane fractions. Since the activation of protein kinase C is known to block phosphoinositol hydrolysis, hippocampal sympathetic ingrowth “normalization” of phosphoinositol hydrolysis may result from a reduction in protein kinase expression in hippocampal sympathetic ingrowth membranes.

Blockade of pilocarpine-induced cerebellar phosphoinositide hydrolysis with metabotropic glutamate antagonists: evidence for an indirect control of granule cell glutamate release by muscarinic agonists

The ability in vivo of the muscarinic agonist, pilocarpine, to increase phosphoinositol (PI) hydrolysis in lithium pretreated rats was investigated by measuring the accumulation of [ 3H]inositol phosphates (IP). As expected, 20 mg/kg s.c. pilocarpine, a muscarinic agonist, increased PI hydrolysis in the striatum, frontal cortex and hippocampus. Somewhat surprisingly, an increase in IP was also found in the cerebellar homogenates. In all four tissues the pilocarpine-induced effect could be completely inhibited by pretreatment with the muscarinic antagonist scopolamine (1.2 mg/kg i.p.). It was also found that the cerebellar but not the hippocampal pilocarpine-induced rise in PI hydrolysis could be blocked by the metabotropic glutamate (mGlu) receptor antagonist, LY341495 (100 nmol, i.c.v.). The same dose of LY341495 was found to also block both the cerebellar and hippocampal increase in IP formed by stimulation with the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (1 μmol, i.c.v.). Given this data and the current information on the distribution of muscarinic and mGlu receptors in the cerebellum, it is suggested that these results may be a reflection of pilocarpine acting at M 2 receptors to indirectly increase glutamate release from parallel fibers by inhibition of γ-aminobutyric acid-releasing Golgi cells.

Role of protein kinase C in angiotensin II-induced constriction of renal microvessels

Role of protein kinase C in angiotensin II-induced constriction of renal microvessels. Although angiotensin II (Ang II) exerts its action through multiple vasomotor mechanisms, the contribution of phosphoinositol hydrolysis products to Ang II-induced renal vasoconstriction remains undetermined. The role of protein kinase C (PKC) in Ang II-induced afferent (AFF) and efferent (EFF) arteriolar constriction was examined using the isolated perfused hydronephrotic rat kidney. Ang II (0.3 nmol/L)-induced EFF constriction was refractory to inhibition of voltage-dependent calcium channels by pranidipine (1 micromol/L, 19 +/- 2% reversal) but was completely reversed by a PKC inhibitor, chelerythrine (1 micromol/L, 96 +/- 2% reversal). Furthermore, direct PKC activation by phorbol myristate acetate (PMA; 1 micromol/L) caused prominent EFF constriction, and this constriction was inhibited by manganese and free calcium medium. In contrast, Ang II-induced AFF constriction was completely abolished by pranidipine (98 +/- 4% reversal) and was partially inhibited by chelerythrine (55 +/- 3% reversal). Although PMA elicited marked AFF constriction, this constriction was insensitive to the calcium antagonist, but was totally inhibited by manganese or free calcium medium. PKC plays an obligatory role in Ang II-induced EFF constriction that requires extracellular calcium entry through nonselective cation channels. In contrast, in concert with our recent findings demonstrating a complete dilation by thapsigargin, Ang II-induced AFF constriction is mainly mediated by inositol trisphosphate (IP3) and voltage-dependent calcium channel pathways, but could not be attributed to the PKC-activated calcium entry pathway (for example, nonselective cation channels). Rather, Ang II-stimulated PKC may cross-talk to the IP3/voltage-dependent calcium channel pathway and could modulate the vasoconstrictor mechanism of the AFF. Thus, the role of PKC during Ang II stimulation differs in AFF and EFF, which may constitute segmental heterogeneity in the renal microvasculature.

Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinin, in its species of origin.

This is the first report of the development of a new radioligand [125I]Bolton-Hunter bufokinin ([125I]BH-bufokinin) and its use in the characterisation of tachykinin receptors in the small intestine of the cane toad, Bufo matrinus. The binding of [125I]BH-bufokinin to toad intestinal membranes was rapid, saturable, of high affinity and to a single population of binding sites with KD 0.57 nM and Bmax 3.1 fmol mg wet weight tissue(-1). The rank order of affinity of tachykinins to compete for [125I]-BH bufokinin binding revealed similarities with that of the mammalian NK1 receptor, being bufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)> or =neuropeptide gamma (NPgamma, 12.4 nM)> or =kassinin (17.8 nM)>scyliorhinin I (35.3 nM)> or =eledoisin (40.6 nM)> or =carassin (43.2 nM)> or =neurokinin A (NKA, 57.8 nM)> or =neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective agonist senktide was a very weak competitor. The radioligand [125I]neurokinin A showed no specific binding to toad intestinal membranes. In the toad isolated small intestine, the maximum contractile response to bufokinin was over 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. Bufokinin was the most potent agonist (EC501 0.34 nM) and produced a long-lasting contraction. Other tachykinins such as physalaemin, SP and kassinin were also potent contractile agents. The potency values of mammalian and amphibian tachykinins derived from functional studies (pD2) correlated significantly with those from binding assays (pKi). The data for fish and molluscan tachykinins, however, showed poor correlation. Contractions to bufokinin and SP were unaffected by atropine, indomethacin and tetrodotoxin. The highly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 were ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with an EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be coupled to phosphoinositol hydrolysis. In summary, this study provides evidence for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor in the toad small intestine. This is probably not the receptor which mediates contraction to carassin, scyliorhinin II and eledoisin. The study also provides evidence that bufokinin and its receptor play an important physiological role in regulating intestinal motility.

Phosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists

The present report describes the effect of mGluR agonists and antagonists administration on phospholipase C activation by measuring accumulation of [ 3H] inositol monophosphates (IP) in rats pre-labeled with [ 3H] myo-inositol (i.c.v. 24 h pre-treatment). The levels of accumulated [ 3H]IP were then determined from clarified tissue homogenates using ion-exchange chromotography. Following lithium chloride treatment (10 mg/kg, s.c.), (R/S)-3,5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist was found to dose-dependently cause a maximal increase in the levels of [ 3H]IP at 0.3 to 3 μmol/8 μl i.c.v. with lower doses resulting in less efficacious or no responses. This effect was temporal-dependent reaching a plateau at 2 h. The DHPG-induced increases in [ 3H]IP were most pronounced in the hippocampus where a 3- to 5-fold increase above vehicle was consistently found, but significant approximately 2-fold increases were also seen in the cerebellum, striatum and frontal cortex. The mixed group I and II agonist, (1S,3R)-1-aminocyclopentane- trans-1,3-dicarboxylic acid (1S,3R- t-ACPD), similarly resulted in dose-dependent increases in [ 3H]IP levels with doses of 1 to 3 μmol i.c.v. Furthermore, this effect was enantiomer specific since the less active 1R,3S- t-ACPD failed to alter phosphoinositol hydrolysis. Administration of the selective mGluR5 agonist (R/S)-2-chloro-5-hydroxyphenyl-glycine (CHPG) resulted in a dose-dependent increase in hippocampal but not cerebellar levels of [ 3H]IP, consistent with the receptor distribution of the two group I mGluRs. The Group II agonist LY354740 (1S,2S,5R,6S-2-aminobicycl[3.1.0]hexane-2,6-dicarboxylate monohydrate) and the group III agonist L-AP 4 (L-(+)-2-amino-4-phosphonobutyric acid) failed to alter the levels of [ 3H]IP. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid) is a nM potent Group II antagonist. However, LY341495 has also been found to have μM potency in inhibiting mGluR1 and 5. The stimulation of [ 3H]PI hydrolysis by 1 μmol DHPG was dose-dependently blocked by co-administration of the mGluR antagonists, LY341495 at doses that are constant with an interaction at Group I mGluR's. Taken together these results suggest that stimulation of group I mGluRs results in measurable increases in PI hydrolysis in vivo. This method could be quite useful in determining the doses and routes of administration of agonists and antagonists that are required to interact with group I mGluRs.

An investigation of the possible interaction of clomethiazole with glutamate and ion channel sites as an explanation of its neuroprotective activity.

The activity of the neuroprotective agent clomethiazole at glutamate and ion channel sites has been investigated. Dizocilpine (3.25 mg/kg intraperitoneally) provided almost total protection against the damage produced by infusion of N-methyl-DL-aspartate (NMDLA; 75 micrograms) into the right hippocampus. In contrast, clomethiazole (96 mg/kg intraperitoneally) was without effect. Using ligand binding techniques, no evidence was found for clomethiazole interacting with NMDA, AMPA or sigma binding sites. Clomethiazole did inhibit the stimulatory effect of the metabotropic glutamate receptor agonist 1S3R-aminocyclopentone-1,3-dicarboxylic acid (ACPD) on phosphoinositol hydrolysis, but only at a concentration of 10(-3) M, which is unlikely to have functional relevance. Clomethiazole was also without effect on ligand binding to Ca2+ channels (N- or L- type), Na+ channels or ATP-sensitive K+ channels. Potentiation of GABA function therefore remains the most plausible explanation for the neuroprotective activity of clomethiazole.

PLC-γ activation is required for PDGF-βR-mediated mitogenesis and monocytic differentiation of myeloid progenitor cells

To investigate the molecular mechanisms mediating hematopoietic cell differentiation and mitogenesis by activation of the platelet-derived growth factor beta receptor (PDGF-betaR), the wild type PDGF-betaR (PDGF-betaRWT) and tyrosine to phenylalanine mutants of the PDGF-betaR, including F751, F966, F970, F1009, F1021 and F1009/F1021 were overexpressed in FDC-P2 myeloid progenitor cells by retroviral-mediated gene transfer. Stimulation of PDGF-betaRWT and F966, F970 and F1009 infectants with PDGF-BB led to the increased expression of monocytic differentiation markers. In contrast, activation of PDGF-betaR in the parental line or the F1021 or F1009/F1021 mutant infectants failed to induce monocytic differentiation. PDGF-BB stimulation of PDGF-betaRWT, F751, F966, F970 and F1009 infectants led to pronounced DNA synthesis, whereas F1021 and F1009/F1021 infectants did not reveal any increase in mitogenesis when compared to that of the FDC-P2 line. While PDGF stimulation of FDC-P2 cells overexpressing PDGF-betaRWT led to a pronounced increase in inositol phosphate formation due to phospholipase C-gamma (PLC-gamma) activation, PDGF-BB induced phosphoinositol hydrolysis was completely abolished in the F1021 and F1009/F1021 infectants. GF 109203X, a specific inhibitor of protein kinase C (PKC) activation, fully blocked PDGF-betaR-mediated monocytic differentiation and mitogenesis. Taken together, these results suggest that stimulation of the PDGF-betaR signaling pathway can mediate monocytic differentiation when PDGF-betaR is expressed at sufficient levels and that activation of PLC-gamma and PKC plays a pivotal role in PDGF-betaR-mediated differentiation and mitogenesis in FDC-P2 cell system.

Effect of hippocampal sympathetic ingrowth and cholinergic denervation on hippocampal phospholipase C activity and G-protein function

Following cholinergic denervation of the hippocampal formation, via medial septal lesions, peripheral noradrenergic fibers, originating from the superior cervical ganglion, grow into the hippocampus. In previous studies, we have found that hippocampal sympathetic ingrowth and cholinergic denervation alone (animals with concurrent medial septal lesions and superior cervical ganglionectomy) alter phosphoinositide turnover and muscarinic cholinergic receptors in such a way as to suggest an alteration in coupling between the muscarinic cholinergic receptors and phosphoinositol turnover. To test this hypothesis we examined the effect of hippocampal sympathetic ingrowth and cholinergic denervation on phospholipase C activity, G-protein function and the whole receptor complex by measuring the amount of phosphoinositide hydrolysed in hippocampal membranes of the rat. Neither hippocampal sympathetic ingrowth nor cholinergic denervation was found to alter phospholipase C activity when activated by increasing concentrations of Ca 2+. In dorsal hippocampus, cholinergic denervation, when compared to hippocampal sympathetic ingrowth and controls, was found to decrease the amount of phosphoinositol hydrolysed when stimulated with the GTP analog, guanosine-5′- O-(3-thiotriphosphate). When guanosine-5′- O-(3-thiotriphosphate) plus carbachol (1 mM) was utilized to stimulate the entire receptor complex, phosphoinositol hydrolysis was found to be decreased in the cholinergic denervation group as compared to both hippocampal sympathetic ingrowth and control groups. This effect was maximum at 3 μM guanosine-5′- O-(3-thiotriphosphate). These results suggest that both hippocampal sympathetic ingrowth and cholinergic denervation affect the efficiency of coupling between the muscarinic cholinergic receptors and phosphoinositol turnover, with cholinergic denervation decreasing and hippocampal sympathetic ingrowth “normalizing” efficiency. Further, they suggest that the G-protein is the site at which hippocampal sympathetic ingrowth and cholinergic denervation mediate their effects. The results of these experiments are also discussed within the context of recent findings demonstrating G-protein abnormalities in Alzheimer's disease.

Serotonin 5-HT2 receptor activation potentiates N-methyl-D-aspartate receptor-mediated ion currents by a protein kinase C-dependent mechanism.

Modulation of N-methyl-D-aspartate (NMDA) receptor-mediated ion currents by serotonin was investigated with a two-electrode voltage clamp technique in Xenopus oocytes injected with rat brain RNA. After a 1-min application of 200 nM serotonin a transient potentiation of the NMDA receptor-mediated ion currents was observed. The serotonin-induced enhancement was mimicked by the protein kinase C activators 1-oleoyl-2-acetyl-sn-glycerol (100 microM) and phorbol 12-myristate 13-acetate (10 nM), whereas the inactive phorbol ester 4-alpha-phorbol 12-myristate 13-acetate (10 nM) had no effect. From these observations it was concluded that protein kinase C was involved in the enhancement of NMDA-induced currents. In agreement with this conclusion, it was found that the serotonin effect was inhibited by the protein kinase C inhibitors sphingosine (1 microM) or staurosporine (1 microM) added 20 min before NMDA application and by oocyte injection of protein kinase C (PKC)-inhibitor peptide (500 ng/oocyte) 1 hr prior to recordings. The serotonin receptor involved was identified as a 5-HT2 receptor subtype by the finding that 200 nM of the selective 5-HT2 receptor agonist alpha-methyl-5-hydroxytryptamine mimicked the potentiation of NMDA-induced ion currents by serotonin. Furthermore, the observed potentiation was significantly reduced by co-application of serotonin with 100 microM of the selective 5-HT2 receptor antagonist ketanserin. These results indicate that 5-HT2 receptors enhance NMDA receptor function via phosphoinositol hydrolysis and subsequent stimulation of PKC.