Stable GTP Analogue Research Articles

The High Affinity Dopamine D2 Receptor Agonist MCL-536: A New Tool for Studying Dopaminergic Contribution to Neurological Disorders.

The dopamine D2 receptor exists in two different states, D2high and D2low; the former is the functional form of the D2 receptor and associates with intracellular G-proteins. The D2 agonist [3H]MCL-536 has high affinity for the D2 receptor (Kd 0.8 nM) and potently displaces the binding of (R-(-)-N-n-propylnorapomorphine (NPA; Ki 0.16 nM) and raclopride (Ki 0.9 nM) in competition binding assays. Here, we further characterize [3H]MCL-536. [3H]MCL-536 was metabolically stable, with about 75% of the compound remaining intact after 1 h incubation with human liver microsomes. Blood-brain barrier penetration in rats was good, attaining at 15 min a % injected dose per gram of wet tissue (%ID/g) of 0.28 in males versus 0.42 in females in the striatum. Specific uptake ratios ([%ID/g striatum]/[%ID/g cerebellum]) were stable in males during the first 60 min and in females up to 15-30 min. The D2-rich striatum exhibited the highest uptake and slowest washout compared to D2-poor cortex or cerebellum. In peripheral organs, uptake peaked at 15 min but declined to baseline at 60 min, indicating good clearance from the body. In vitro autoradiography on transaxial and coronal brain sections showed specific binding of [3H]MCL-536, which was abolished by preincubation with D2/D3 ligands sulpiride, NPA, and raclopride and in the presence of the stable GTP analogue guanylylimidodiphosphate. In amphetamine-sensitized animals, striatal binding was higher than in controls, indicating specificity for the D2high receptor state. [3H]MCL-536's unique properties make it a valuable tool for research on neurological disorders involving the dopaminergic system like Parkinson's disease or schizophrenia.

Impact of G12 Mutations on the Structure of K-Ras Probed by Ultraviolet Photodissociation Mass Spectrometry.

Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5'-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation.

Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor.

Adenosine A1 receptors in human brain and transfected CHO cells: Inhibition of [3H]CPFPX binding by adenosine and caffeine

In vivo imaging of adenosine function has become feasible with the specific A1 adenosine receptor ligand [18F]CPFPX and positron emission tomography (PET). It is, however, still an open question whether [18F]CPFPX is displaceable by endogenous adenosine, which would allow to detect activity-dependent adenosine release in vivo. We used the tritiated analog of [18F]CPFPX, [3H]CPFPX, to quantify A1 adenosine receptors (A1AR) in grey matter tissue homogenates of four human brains and A1AR transfected Chinese hamster ovary cells, respectively. Saturation binding experiments in the presence of a stable GTP analog revealed a dissociation constant (KD) of 2.4±0.5nM. The unselective endogenous A1AR agonist adenosine and the antagonist caffeine displaced specific [3H]CPFPX binding completely at high doses. Concentrations sufficient to inhibit 50% of binding (IC50) were 6.9±2.7μM for adenosine and 148±15.4μM for caffeine. Respective inhibition constants (Ki) were 2.8±0.9μM and 61.4±11.2μM.The present report supports the possibility of studying acute effects of adenosine and caffeine in vivo with [18F]CPFPX and PET. Pathophysiological conditions like hypoxia which increase endogenous adenosine concentrations several folds might interfere with in vivo [18F]CPFPX binding. Caffeine intake previous to the investigation should be considered as a confounding factor regarding the determination of receptor densities with [18F]CPFPX and PET.

Agonist binding fraction of dopamine D 2/3 receptors in rat brain: A quantitative autoradiographic study

There has arisen considerable interest in the study of dopamine D 2/3 agonist binding sites by positron emission tomography (PET), based on the claim that agonist sites represent a functional subset of the total number of sites labeled by more conventional antagonist ligands. To test the basis of this claim, we used quantitative autoradiography to measure the abundance of binding sites of a dopamine D 2/3 agonist ([ 3H]NPA) and an antagonist ([ 3H]raclopride) in cryosections of rat brain. Saturation binding studies revealed that the B max for [ 3H]NPA was nearly identical to that of [ 3H]raclopride in dorsal brain regions, but was 25% less in the ventral striatum and 56% less in the olfactory tubercle. We also tested the displacement of the two ligands by the hallucinogen LSD, which is known to have dopamine agonist properties. Whereas displacement of [ 3H]raclopride by increasing LSD concentrations was monophasic, displacement of [ 3H]NPA was biphasic, suggesting an action of LSD via a subset of dopamine D 2/3 agonist binding sites. Addition of the stable GTP analogue Gpp(NH)p to the medium abolished 90% of the [ 3H]NPA binding, and increased [ 3H]raclopride binding by 10%, with a shift to the right in the LSD competition curve, suggesting retention of endogenous dopamine in washed cryostat sections. Thus [ 3H]NPA and [ 3H]raclopride binding sites have nearly identical abundances in rat dorsal striatum, but are distinct in the ventral striatum, and with respect to their displacement by LSD.

Differential interactions of G-proteins and adenylyl cyclase with nucleoside 5′-triphosphates, nucleoside 5′-[γ-thio]triphosphates and nucleoside 5′-[β,γ-imido]triphosphates

The regulatory G-proteins of adenylyl cyclase (AC), G i and G s, are not only activated by GTP and the stable GTP analogs, guanosine 5′-[γ-thio]triphosphate (GTPγS) and guanosine 5′-[β,γ-imido]triphosphate (GppNHp), but also by hypoxanthine, xanthine, uracil and cytidine nucleotides. The latter nucleotides were previously used to analyze distinct active G-protein states. Surprisingly, recent studies have shown that inosine 5′-[γ-thio]triphosphate and uridine 5′-[γ-thio]triphosphate can also inhibit AC directly. Therefore, we systematically compared the interactions of nucleoside 5′-triphosphates (NTPs), nucleoside 5′-[γ-thio]triphosphates (NTPγSs) and nucleoside 5′-[β,γ-imido]triphosphates (NppNHps) with G i, G s and AC. NTPγSs exhibited up to 26,000-fold higher affinity for G-proteins than NTPs and NppNHps. NTPγSs were up to 150-fold more potent direct AC inhibitors than NTPs and NppNHps. G-proteins exhibited striking preference for guanine nucleotides compared to other purine and pyrimidine nucleotides, whereas base-selectivity of various ACs, particularly the purified catalytic subunits C1·C2, was rather poor. GTP, GTPγS and GppNHp exhibited much higher selectivity for G-proteins relative to AC than all other purine and pyrimidine nucleotides. We have energetically characterized the interactions of purine and pyrimidine nucleotides with AC in silico, constructing pharmacophore models that correlate well with experimental affinities and have elucidated specific amino acid residues with greatest influence on nucleotide binding. Collectively, both G-proteins and ACs bind purine and pyrimidine nucleotides, with G-proteins showing much higher base-selectivity than AC. Thus, direct inhibitory effects of nucleotides on AC should be understood and considered when probing distinct active G-protein states with non-guanine nucleotides.

Differences in type II, IV, V and VI adenylyl cyclase isoform expression between rat preadipocytes and adipocytes

Adenylyl cyclase catalytic activity is low in preadipocyte membranes when compared to adipocytes. Under conditions promoting inhibition of adipocyte adenylyl cyclase activity by Gpp(NH)p, a stable GTP analog, a paradoxical increase in preadipocyte adenylyl cyclase activity was obtained. In order to explain this contradiction, expression of types II, IV, V and VI adenylyl cyclase isoforms was compared in adipocytes and undifferentiated preadipocytes both by western blots and by a semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) assay. Type II, IV, V and VI mRNAs and proteins were present in both adipocytes and preadipocytes. However, in undifferentiated preadipocytes, expression of type II mRNA and protein were significantly higher whereas expression of type IV, V and VI adenylyl cyclase mRNAs and proteins were significantly weaker than in adipocytes. In late differentiated preadipocytes, the adenylyl cyclase subtype mRNA expression pattern was intermediary between the undifferentiated and the full differentiation states except for type IV which remained weakly expressed. Moreover, one of the representative r egulators of G -protein s ignaling (RGS protein), RGS4, was less expressed in undifferentiated preadipocyte membranes and cytosol extracts, which contrasts with adipocytes where RGS4 is clearly expressed. Thus, the preferential expression of type II adenylyl cyclase (G βγ subunit-stimulated) in preadipocytes might explain why Gpp(NH)p elicits stimulation of adenylyl cyclase under conditions designed to promote inhibition. Conversely, the preferential expression of type V and VI adenylyl cyclases and the slightly higher expression of type IV adenylyl cyclase in adipocytes could contribute to explain the elevated total catalytic activity observed in mature fat cells compared to their precursor cells.

Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation.

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation.

Formyl Peptide Receptor Signaling in HL-60 Cells through Sphingosine Kinase

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.

Subtype-specific stimulation of [ [formula omitted]]GTPγS binding by recombinant α 2-adrenoceptors

We measured agonist-stimulated binding of the stable GTP-analog guanosine-5′- O-(3-[ 35 S ]thiotriphosphate) ([ 35 S ]GTPγS) as a functional assay to monitor G-protein activation by recombinant human α 2-adrenoceptor subtypes α 2A, α 2B and α 2C. (−)-Noradrenaline was a full agonist in all receptors. Dexmedetomidine, UK14,304, clonidine and oxymetazoline showed subtype-selectivity in efficacy. Dexmedetomidine was a full agonist at α 2B and a partial agonist at α 2A; UK14,304 was a full agonist at α 2A and a partial agonist at α 2B. Clonidine and oxymetazoline were weak partial agonists at the α 2B-subtype, but appeared inactive at α 2A and α 2C. The [ 35 S ]GTPγS binding assay provides functional information on pertussis toxin-sensitive G-protein activation, complementing radioligand binding assays and conventional second messenger assays.

Interactions of Phosducin with the Subunits of G-Proteins: BINDING TO THE α AS WELL AS THE βγ SUBUNITS

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Role of fluidity of membranes on the guanyl nucleotide-dependent binding of cholecystokinin-8S to rat brain cortical membranes

The binding of [ 3H]cholecystokinin octapeptide (sulphated) ([ 3H]CCK-8S), an agonist of the cholecystokinin receptors, to rat cortical membranes was fast, specific and saturable, with pH optimum at 6.5–7.0. The divalent cations Mg 2+ and Ca 2+ clearly enhanced [ 3H]CCK-8S binding, whereas the monovalent cations Na + and K + were inhibitors. Inactivation of the ligand binding ability of these membranes was dependent on the incubation temperature and corresponding τ 1 2 values were 11 days at 4°, 12 hr at 21°, 154 min at 30° and 51 min at 37°, which revealed the apparent activation energy of this process to be 130 ± 4 kJ/mol. Scatchard analysis of the saturation curves of [ 3H]CCK-8S binding was best described by a one site binding model with a K d = 0.63 ± 0.18 M and a maximum binding of 32 ± 2 fmol/mg protein. The stable GTP analogue guanosin-5′-O-(3-thiotriphosphate) (GTPγS) decreased the affinity of [ 3H]CCK-8S binding only up to 2-fold without significant influence on maximal binding. Modulation of membrane properties by different detergents revealed that only in the case of digitonin (0.03–0.04%) did the GTP-dependence of [ 3H]CCK-8S binding considerably increase without significant influence on the ligand binding properties in the absence of GTPγS. Other detergents studied (sodium cholate, sodium deoxycholate, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), sucrose monolaurate, series Triton X and Tween) either had little influence on GTPγS-dependence of [ 3H]CCK-8S binding or inactivated the receptor. Parallel studies of fluorescent polarization of diphenylhexatriene (DPH) in rat cortical membranes indicated that digitonin was the only detergent which at low concentrations caused a rapid increase in membrane fluidity and thereafter stabilized it at a certain level. Other detergents studied had only moderate influence on membrane fluidity (CHAPS, cholate, deoxycholate) or caused fast and continuous increase of membrane fluidity (Triton X-100, Tween 80). These data together point to the essential influence of the fluidity of membranes on the regulation of the interactions between G proteins and CCK receptors in rat cortical membranes. Under standard experimental conditions (temperature lower than 30°), the CCK receptor-G protein complex is active for quantitative characterization of the receptors, but the membranes are too rigid for natural communication and regulation.

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists.

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.

Myosin light-chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade.

The aim of this study was to investigate how insulin secretion is controlled by phosphorylation of the myosin light chain (MLC). Ca2+-evoked insulin release from pancreatic islets permeabilized with streptolysin O was inhibited by different monoclonal antibodies against myosin light-chain kinase (MLCK) to an extent parallel to their inhibition of purified MLCK. Anti-MLCK antibody also inhibited insulin release caused by the stable GTP analog guanosine 5'-O-(3-thiodiphosphate), even at a substimulatory concentration (0.1 microM) of Ca2+. Free Ca2+ increased MLC peptide phosphorylation by beta-cell extracts in vitro. In contrast to the phosphorylation by purified MLCK or by calmodulin (CaM) kinase II, the activity partially remained with the beta-cell under nonstimulatory Ca2+ (0.1 microM) conditions. The MLCK inhibitor ML-9 inhibited the activity in the beta-cell with both substimulatory and stimulatory Ca2+, whereas KN-62, an inhibitor of CaM kinase II, only exerted an influence in the latter case. ML-9 decreased intracellular granule movement in MIN6 cells under basal and acetylcholine-stimulated conditions. We propose that MLC phosphorylation may modulate translocation of secretory granules, resulting in enhanced insulin secretion.

Influence of fluoxetine and p-chloroamphetamine on the somatostatin receptor–adenylyl cyclase system in the rat frontoparietal cortex

There is evidence that suggests a reciprocal functional link between the serotonergic and the somatostatinergic system in the rat frontoparietal cortex. However, to date, the role of endogenous 5-hydroxytryptamine (serotonin) on the regulation of the somatostatin (SS) receptor–adenylyl cyclase (AC) system remains unclear. In the present study, the administration of fluoxetine (10 mg/kg i.p.), a 5-hydroxytryptamine uptake inhibitor in a single dose or administered daily for 14 days increased the number of specific [ 125I]Tyr 11-SS receptors, with no change in the receptor affinity, in rat frontoparietal cortical membranes. However, the capacity of SS to inhibit forskolin (FK)-stimulated AC activity in these membranes was lower than in the control groups. The ability of the stable GTP analogue 5′-guanylylimidodiphosphate (Gpp(NH)p) to inhibit FK-stimulated AC activity in frontoparietal cortical membranes was also decreased in rats acutely and chronically treated with fluoxetine. p-Chloroamphetamine (5 mg/kg i.p.), which leads to a lasting reduction of 5-hydroxytryptamine innervation, administered on days 1, 3 and 5 and the rats sacrificed 1 or 3 weeks after the first injection, decreased the number of SS receptors without changing the receptor affinity. In this experimental group, SS also caused a significantly lower inhibition of FK-stimulated AC activity. p-Chloroamphetamine had no effect on the ability of Gpp(NH)p to inhibit FK-stimulated AC activity in frontoparietal cortical membranes at all the time periods studied. The present results suggest that under normal circumstances some SS receptors are under a tonic stimulatory control through the serotonergic system.

Characterization and purification from bovine neutrophils of a soluble guanine-nucleotide-binding protein that mediates isozyme-specific stimulation of phospholipase C beta2.

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.

5-Hydroxytryptamine decreases somatostatin receptors and somatostatin-responsive adenylyl cyclase in rat pancreatic acinar membranes

Pretreatment of pancreatic acini with 5-hydroxytryptamine (5-HT) reduced the binding of the labeled somatostatin (SS) analogue 125I-Tyr 3-SMS to pancreatic acinar membranes. This effect was dependent of the dose of 5-HT used and length of pretreatment. This inhibitory effect of 5-HT was abolished when pancreatic acini were pretreated with 5-HT in the presence of the 5-HT 1P receptor-antagonist 5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP). Pretreatment of pancreatic acini with 5-HT reduced the inhibition by the stable SS analogue SMS 201-995 of basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity in pancreatic acinar membranes. There was no statistical difference established between IC 50 values for the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp(NH)p) which inhibits ligand binding to SMS receptors in controls and in 5-HT treated pancreatic cells, respectively. In addition, no significant differences were seen in the level of Gi proteins in the control and 5-HT treated panceatic acini. These data suggest that the decrease of the number of 125I-Tyr 3-SMS receptors, would explain the decreased sensitivity of AC to SMS 201-995 in membranes from 5-HT-pretreated acini.

Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors.

The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C. botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], a substrate and cofactor for PLC and PLD, respectively, were not altered. These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme. In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.

Histamine H 1-Receptors Modulate Somatostatin Receptors Coupled to the Inhibition of Adenylyl Cyclase in the Rat Frontoparietal Cortex

Puebla, L., A. OcaÑa and E. Arilla. Histamine H 1-receptors modulate somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex. Peptides 18(10) 1569–1576, 1997.—Since exogenous histamine has been previously shown to increase the somatostatin (SS) receptor-effector system in the rat frontoparietal cortex and both histamine H 1-receptor agonists and SS modulate higher nervous activity and have anticonvulsive properties, it was of interest to determine the participation of the H 1-histaminergic system in this response. The intracerebroventricular (i.c.v.) administration of the specific histamine H 1-receptor agonist 2-pyridylethylamine (PEA) (10 μg) to rats 2 h before decapitation increased the number of SS receptors (599 ± 40 vs 401 ± 31 femtomoles/mg protein, p< 0.01) and decreased their apparent affinity for SS (0.41 ± 0.03 vs 0.26 ± 0.02 nM, p < 0.01) in rat frontoparietal cortical membranes. No significant differences were seen for the basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activities in the frontoparietal cortex of PEA-treated rats when compared to the control group. In the PEA group, however, the capacity of SS (10 −4 M) to inhibit basal and FK (10 −5 M)-stimulated AC activity in frontoparietal cortical membranes was significantly higher than in the control group (34 ± 1% vs 20 ± 2%, p < 0.001). The ability of low concentrations of the stable GTP analogue 5′-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity in frontoparietal cortical membranes was similar in the PEA-treated and control animals. These results suggest that the increased SS-mediated inhibition of AC activity in the frontoparietal cortex of PEA-treated rats may be due to the increase of the number of SS receptors induced by PEA. Pretreatment with the H 1-receptor antagonist mepyramine (30 mg/kg, intraperitoneally (IP) prevented the PEA-induced changes in SS binding and SS-mediated inhibition of AC activity. Mepyramine (30 mg/kg, IP) alone had no observable effect on the somatostatinergic system. The in vitro addition of PEA or mepyramine to frontoparietal cortical membranes obtained from untreated rats did not affect the SS binding parameters. Altogether, these results suggest that the H 1-histaminergic system modulates the somatostatinergic system in the rat frontoparietal cortex.

Signalling properties of lysophosphatidic acid in primary human skin fibroblasts: role of pertussis toxin-sensitive GTP-binding proteins.

We have investigated the signalling properties of the naturally occurring intercellular signalling molecule lysophosphatidic acid (LPA) in primary human skin fibroblasts. LPA stimulated phospholipase C activity resulting in the formation of inositol 1,4,5-trisphosphate (IP3) which was accompanied by a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was subject to homologous desensitisation but not to heterologous desensitisation by sphingosine-1-phosphate. The half-maximal effect of LPA on the rise in [Ca2+]i was attained at 7-20 nM. IP3 formation and Ca2+ mobilisation were highly pertussis toxin (PTX)-sensitive (100% and 75%, respectively). LPA also inhibited forskolin-stimulated formation of cAMP, which was partially reversed (51%) when fibroblasts were pretreated with PTX. To directly test the involvement of guanine nucleotide-binding regulatory proteins (G proteins), LPA-induced binding of the stable GTP analogue GTP gamma S was measured. LPA induced an increase in GTP gamma S binding, which was completely inhibited by PTX, implicating the involvement of Gi-type G proteins in LPA signalling. Furthermore, LPA increased DNA synthesis and cell proliferation. Finally, LPA induced the migration of human skin fibroblasts, which in conjunction with the stimulation of cell growth strengthens the presumed involvement of LPA in wound healing and tissue regeneration. Both effects (cell growth and migration) were almost completely PTX-sensitive. Overall, these investigations in primary cultures of human skin fibroblasts confirm and extend our knowledge about LPA signalling, suggesting a pivotal role of receptor coupled activation of Gi-type proteins at least in this cell type.