Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Activation of Opioid and Muscarinic Receptors Stimulates Basal Adenylyl Cyclase but Inhibits Ca2+/Calmodulin- and Forskolin-Stimulated Enzyme Activities in Rat Olfactory Bulb

Abstract: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca²⁺ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu⁵]Enkephalin and selective δ- and μ-, but not κ-, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu⁵]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca²⁺/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb δ- and μ-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.     

Phospholipid metabolism is required for M<Subscript>1</Subscript> muscarinic inhibition of N-type calcium current in sympathetic neurons

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca²⁺ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein G_q and identified phospholipase C (PLC), phospholipase A₂ (PLA₂), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP₂) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M₂/M₄ muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M₁ muscarinic receptors (M₁Rs), G_q, PLC, PLA₂, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA₂ cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M₁Rs, G_q, PLC, PIP₂, PLA₂, and AA for N-current inhibition.Abbreviations AA arachidonic acid - BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - DAG diacylglycerol - DEDA 7,7-dimethyleicosadienoic acid - ETYA 5,8,11,14-eicosatetraynoic acid - FPL FPL 64176 - IP₃ inositol-1,4,5-trisphosphate - L-channel L-type calcium channel - L-current L-type calcium current - LPA lysophosphatidic acid - M₁R M₁ muscarinic receptor - N-channel N-type calcium channel - N-current N-type calcium current - NMN nimodipine - OAG 1-(cis-9-octadecenoyl)-2-acetyl-sn-glycerol - OPC oleoyloxyethyl phosphorylcholine - Oxo-M oxotremorine methiodide - PIP₂ phosphatidylinositol-4,5-bisphosphate - PLC phospholipase C - PLA₂ phospholipase A₂ - PTX pertussis toxin - SCG superior cervical ganglion     

M1 Muscarinic Receptors Contribute to, whereas M4 Receptors Inhibit, Dopamine D1 Receptor-Induced [3H]-Cyclic AMP Accumulation in Rat Striatal Slices

In rat striatal slices labelled with [³H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [³H]-AMP ([³H]-cAMP) accumulation induced by the dopamine D₁ receptor agonist SKF-81297 (1 μM; 177±13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72±3%, IC₅₀ 0.30±0.06 μM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M₁ receptors, reduced the effect of SKF-81297 by 40±7% (IC₅₀ 251±57 pM) and enhanced the inhibitory action of a submaximal (1 μM) concentration of carbachol (69±4% vs. 40±7% inhibition, IC₅₀ 386±105 pM). The toxin MT-1, agonist at M₁ receptors, stimulated [³H]-cAMP accumulation in a modest but significant manner (137±11% of basal at 400 nM), an action additive to that of D₁ receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D₁ receptor-induced [³H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M₄ receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M₁ receptors and PKC stimulation.     

Discovery of N-sulfonyl-7-azaindoline derivatives as potent,orally available and selective M4 muscarinic acetylcholine receptor agonists

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1–10 mg/kg, po).     

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M₁ receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [³H]N-methylscopolamine binding to cloned muscarinic M₁ and M₄ receptors, and reduced binding to M₅ subtype with lower affinity, while they reversibly inhibited the binding of [³H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M₁ receptors at a site distinct from the classical one, and to have affinity for some -adrenoceptors.     

Amitriptyline reduces olfactory neuron adenylyl cyclase activity

Adenylyl cyclase plays an important role in olfactory signaltransduction. Recently, a novel type III adenylyl cyclase hasbeen localized in olfactory neurons (Pfeuffer et al., 1989;Bakalyar and Reed, 1990). Because amitriptyline (AMI), a tricyclicantidepressant, appears to have an inhibitory effect on adenylylcyclase activity in other in other neuronal tissue (Yamaokaet al., 1988; Wong et al., 1991), we measured the effect ofAMI on forskolin-stimulated adenylyl cyclase activity in membranepreparations of olfactory mucosa from adult rats. In the presenceof 5'-guanylyl-imidodiphosphate, AMI (0.5–8.0 µM)inhibited forskolin-stimulated adenylyl cyclase activity ina dose-dependent manner. To determine whether this effect wasspecific for olfactory neurons, as opposed to other cells inthe olfactory epithelium, rats were unilaterally bulbectomizedin order to reduce selectively the number of olfactory neuronson the side ipsilateral to the bulbectomy. In membrane preparationsfrom unilaterally bulbectomized animals we saw significantlylower adenylyl cyclase activity in ipsilateral olfactory mucosa,compared with adenylyl cyclase activity from non-bulbectomizedmucosa. These results indicate that AMI inhibition of adenylylcyclase activity is primariy localized in olfactory neurons.     

Phorbol Esters Increase GTP-Dependent Adenylate Cyclase Activity in Rat Brain Striatal Membranes

Abstract: 4β-Phorbol 12-myristate 13-acetate (PMA), added to a lysed mitochondrial fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of ～30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PMA, respectively. The PMA stimulation is GTP dependent, reaching a maximum of ～60% at 50 μ.M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5′-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalin, and R(-)N⁶-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5′-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by l-α-phosphatidyl-l-serine. Like PMA, 4β-phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4β-phorbol and 4β-phorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.     

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.     

Characterization of Muscarinic Acetylcholine Receptors in Cultured Bovine Aortic Endothelial Cells

Abstract

The binding characteristics of [³H]quinuclidinyl benzilate ([³H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [³H]QNB bound to endothelial cell membranes with high affinity (k_D = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (K_i) were determined by displacement of bound [³H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M₃ antagonist) > pirenzepine (M₁ antagonist) > AFDX-116 (M₂ antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M₁ agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M₃ and M₁ subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [³H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [³H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.     

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC₅₀ values for 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10⁻⁵ M), quisqualate was the most potent in all the brain areas (range 10⁻⁷−10⁻⁶ M), except the cerebellum (10⁻⁵ M), ibotenate was the most potent in the striatum (range 10⁻⁶ M) and the least potent in the cerebral cortex and hippocampus (range 10⁻⁴ M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45%, respectively, without differences between brain areas. On the other hand, the antagonistic effects ofl-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75%) and the lowest in the cerebellum (25%). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.     

Characterization of structure and activity of garlic peroxidase (POX<Subscript>1B</Subscript>)

Structural characterization and study of the activity of new POX_1B protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure–function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX_1B exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k _cat and K _M values were 411 and 400 mM⁻¹ s⁻¹ for 3,3′,5,5′-tetramethylbenzidine and 2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX_1B was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX_1B showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX_1B) is suitable for the monitoring of different analytes and biocatalysis.     

The effect of GTP on the aluminum fluoride- and forskolin-activated adenylyl cyclase from human embryonic kidney 293 cells

GTP has been shown to inhibit AlF₄⁻-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg²⁺ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF₄⁻-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg²⁺, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn²⁺ ions stimulated AlF₄⁻- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg²⁺. The inhibition of AlF₄⁻-stimulated cyclase by GTP was still observed with Mn²⁺, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn²⁺ and Mg²⁺ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF₄⁻-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF₄⁻ or under basal conditions was observed to be somehow different in the presence of Mn²⁺ than in the presence of Mg²⁺ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF₄⁻. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF₄⁻-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF₄⁻.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Differential Requirements of Sodium for Coupling of Cannabinoid Receptors to Adenylyl Cyclase in Rat Brain Membranes

Abstract: Sodium is generally required for optimal inhibition of adenylyl cyclase by G_l/o-coupled receptors. Canna-binoids bind to specific receptors that act like other members of the G_l/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCI ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABA_B receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCI providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low K_m GTPase activity was maximal at 50 mM NaCI in striatal membranes, but was maximal in the absence of NaCI in cerebellar membranes. Assay of [³H]WIN 55212–2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other G_l/o-coupled receptors, because both NaCI and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

C<Subscript>1</Subscript>-/C<Subscript>2</Subscript>-aromatic-imino-glyco-conjugates: experimental and computational studies of binding,inhibition and docking aspects towards glycosidases isolated from soybean and jack bean

Several C₁-imino conjugates of d-galactose, d-lactose and d-ribose, where the nitrogen center was substituted by the salicylidene or naphthylidene, were synthesized and characterized. Similar C₂-imino conjugates of d-glucose have also been synthesized. All the glyco-imino-conjugates, which are transition state analogues, exhibited 100% inhibition of the activity towards glycosidases extracted from soybean and jack bean meal. Among these, a galactosyl-napthyl-imine-conjugate (1c) showed 50% inhibition of the activity of pure α-mannosidase from jack bean at 22 ± 2.5 μM, and a ribosyl-naphthyl-imine-conjugate (3c) showed at 31 ± 5.5 μM and hence these conjugates are potent inhibitors of glycosidases. The kinetic studies suggested non-competitive inhibition by these conjugates. The studies are also suggestive of the involvement of aromatic, imine and carbohydrate moieties of the glyco-imino-conjugates in the effective inhibition. The binding of glyco-imino-conjugate has been established by extensive studies carried out using fluorescence emission and isothermal titration calorimetry. The conformational changes resulted in the enzyme upon interaction of these derivatives has been established by studying the fluorescence quench of the enzyme by KI as well as from the secondary structural changes noticed in CD spectra. All these studies revealed the difference in the binding strengths of the naphthylidene vs. salicylidene as well as galactosyl vs. lactosyl moieties present in these conjugates. The differential inhibition of these glyco-conjugates has been addressed by quantifying the specific interactions present between the glyco-conjugates and the enzyme by using rigid docking studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.