Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Activation of Cl− channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca²⁺ concentration ([Ca²⁺]₀) were examined in freshly isolated rabbit osteoclasts. K⁺ current was suppressed by intracellular and extracellular Cs⁺ ions. In this condition, high [Ca²⁺]₀ evoked an outwardly rectifying current with a reversal potential of about −25 mV. When the concentration of extracellular Cl⁻ ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4′,4-diisothiocyanostilbene-2′,2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl⁻ channels. Cd²⁺ or Ni²⁺ caused a transient activation of the Cl⁻ current in contrast to the sustained activation elicited by Ca²⁺. Intracellular 20 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl⁻ current. Either when the osmolarity of extracellular medium was increased, or when 100 μM cAMP was dissolved in the patch pipette solution, high [Ca²⁺]₀ still elicited the Cl⁻ current, indicating that the divalent cation-induced Cl⁻ current was carried through Ca²⁺-activated Cl⁻ channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl⁻ current, indicating that the activation of Cl⁻ current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl⁻ channels under whole cell clamp. Intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) activated the Cl⁻ current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl⁻ current was mediated through a G protein, and that an increase in [Ca²⁺]_i was critical for the activation of Cl⁻ channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl⁻ current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca²⁺-activated Cl⁻ channels. © 1996 Wiley-Liss, Inc.     

The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation

These studies were designed to compare the effects of nitric oxide (NO) generating compounds with those of several iron containing, compounds which do not generate NO on glutamate receptor function. Stimulation of primary cultures of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainate results in the elevation of intracellular calcium ([Ca²⁺]_i) and cGMP and the release of glutamate. The iron containing compounds, sodium nitroprusside (SNP), potassium ferrocyanide (K₄Fe(CN)₆) and potassium ferricyanide (K₃Fe(CN)₆) decrease the NMDA-induced release of glutamate. SNP is the only compound of the above 3 agents which generates NO. A non-iron, NO generating compound, S-nitroso-N-acetylpenicillamin (SNAP), has no effect on the NMDA-induced glutamate release. Potassium ferrocyanide (Fe II), but not potassium ferricyanide (Fe III), blocks NMDA-induced cGMP elevations after 3 min exposure times. This contrasts with the NO generating compounds (both SNP and SNAP) which elevate cGMP levels. Furthermore, both potassium ferrocyanide (Fe II) and SNP (Fe II) suppress the elevation of [Ca²⁺]_i induced by NMDA but neither potassium ferricyanide (Fe III) nor SNAP are effective in this regard. These effects are also independent of cyanide as another Fe II compound, ferrous sulfate (FeSO₄) is also able to suppress NMDA-induced elevations of [Ca²⁺]_i SNP was unable to suppress kainate receptor functions. Collectively, these results indicate that Fe II, independently of NO, has effects on NMDA receptor function.     

Effects of phosphonium-based ionic liquids on the lipase activity evaluated by experimental results and molecular docking

In this work, the effect of several phosphonium-based ionic liquids (ILs) on the activity of lipase from Burkholderia cepacia (BCL) was evaluated by experimental assays and molecular docking. ILs comprising different cations ([P₄₄₄₄]⁺, [P_444(14)]⁺, [P_666(14)]⁺) and anions (Cl⁻, Br⁻, [Deca]⁻, [Phosp]⁻, [NTf₂]⁻) were investigated to appraise the individual roles of IL ions on the BCL activity. From the activity assays, it was found that an increase in the cation alkyl chain length leads to a decrease on the BCL enzymatic activity. ILs with the anions [Phosp]⁻ and [NTf₂]⁻ increase the BCL activity, while the remaining [P_666(14)]-based ILs with the Cl⁻, Br⁻, and [Deca]⁻ anions display a negative effect on the BCL activity. The highest activity of BCL was identified with the IL [P_666(14)][NTf₂] (increase in the enzymatic activity of BCL by 61% at 0.055 mol·L⁻¹). According to the interactions determined by molecular docking, IL cations preferentially interact with the Leu17 residue (amino acid present in the BCL oxyanion hole). The anion [Deca]⁻ has a higher binding affinity compared to Cl⁻ and Br⁻, and mainly interacts by hydrogen-bonding with Ser87, an amino acid residue which constitutes the catalytic triad of BCL. The anions [Phosp]⁻ and [NTf₂]⁻ have high binding energies (−6.2 and −5.6 kcal·mol⁻¹, respectively) with BCL, and preferentially interact with the side chain amino acids of the enzyme and not with residues of the active site. Furthermore, FTIR analysis of the protein secondary structure show that ILs that lead to a decrease on the α-helix content result in a higher BCL activity, which may be derived from an easier access of the substrate to the BCL active site.     

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI⁻ secretion, their effects on CI⁻ transport and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl⁻ sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca²⁺-sensitive fluorescent probe, fura-2AM were used to assess Cl⁻ transport and [Ca²⁺]_i, respectively. Basal Cl⁻ transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl⁻ channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na⁺/K⁺/2Cl⁻ cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I⁻ > F⁻ ≥ Cl⁻ > Br⁻. DPC inhibited I⁻ influx by ∼50%, F⁻ influx by 80%, Cl⁻ influx by 85%, and Br⁻ influx by 90%. Furosemide significantly inhibited influx of Br⁻ (84%) and Cl⁻ (81%) but not of F⁻ and I⁻. The effects of agents known to alter biological response by increasing [Ca²⁺]_i in other epithelial systems were used to stimulate Cl⁻ transport. Cl⁻ influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca²⁺ ionophore A23187 (2.05 ± 0.40). The Cl⁻ permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl⁻ influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl⁻ permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl⁻ transport by activating the Na⁺/K⁺/2Cl⁻ cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl⁻ channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc.     

The effects of nitrite on chloride regulation in the crayfish Pacifastacus leniusculus Dana (Crustacea: Decapoda)

Summary Nitrite in the external freshwater medium was found to be toxic to Pacifastacus leniusculus Dana (48 h LC₅₀0.7 mM NO ₂ ^– ). It produced significant changes in haemolymph ionic concentration and acid-base status. Exposure to 1.0 mM NO ₂ ^– resulted in a rapid, active accumulation of nitrite in the haemolymph (to 25 mM NO ₂ ^– after 24 h) and caused the partial inhibition of Cl^– uptake. Some reduction in Cl^– efflux rate was seen. In 1.0 mM NO ₂ ^– a rapid depletion of haemolymph [Cl^–] was observed (50 mM decrease in 27 h). Nitrite competitively inhibited active Cl^– uptake (K_m increased from 0.42 to 1.22 mM; K_i=0.45 mM). To achieve Cl^– balance in this medium, depleted crayfish would require a two-fold increase in external [Cl^–]. A lesser decrease in haemolymph [Na⁺] was found while osmotic pressure was relatively unaffected. Haemolymph [HCO ₃ ^– ] showed a significant increase and was accompanied, unexpectedly, by an acidosis. Possible sources of the excess HCO ₃ ^– , perhaps by inhibition of normal Cl^–/HCO ₃ ^– branchial exchange or release from CaCO₃ stores, are discussed. Haemolymph clearance of NO ₂ ^– was slower than uptake as was the restoration of [Cl^–] on recovery in nitrite-free medium.Abbreviations AFWM artificial freshwater medium - BOD biochemical oxygen demand - J _out ^Cl chloride efflux - J _in ^Cl chloride influx - J _in ^Cl chloride influx - J _net ^base net base flux - J _net ^base net base flux - J _in(p) ^Cl passive chloride influx - J _out efflux - LC ₅₀ median lethal concentration - NEDE N-1-Naphthylethylenediamine - SEM standard error of mean - TEP transepithelial potential difference - V _in ^Cl active chloride uptake     

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells’ (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5 mm diameter cattle follicles was cultured without treatment (control), with ODQ (10⁻⁴ M) and 10⁻⁵, 10⁻³ and 10⁻¹ M SNP with or without ODQ for 24 h. Nitrate/nitrite (NO₃⁻/N0₂⁻) concentrations were evaluated by Griess method, progesterone (P₄) and 17β-estradiol (E₂) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P < 0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10⁻⁵ M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P > 0.05), while GC cultured with 10⁻³ and 10⁻¹ M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P < 0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10⁻⁵ and 10⁻³ M SNP with or without ODQ were in the G0/G1 (80–75%) stage and in a lesser proportion (20–25%) in the S + G2/M stage of the cell cycle, while the 10⁻¹ M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10⁻⁵ M SNP increased (P < 0.05) E₂ synthesis and inhibited (P < 0.05) progesterone synthesis. The addition of ODQ reversed (P < 0.05) the stimulatory effect of 10⁻⁵ M SNP treatment on E₂, but not on P₄ synthesis (P > 0.05). These results demonstrated that E₂ synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P₄ while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.     

Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism

Introduction

The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way.

Methods

Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (Δψm) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[³H]glucose (2-[³H]DG). Statistical analysis was performed using the Mann-Whitney U test.

Results

NOC-12 liberates approximately ten times more NO₂^- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ΔΨm and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[³H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[³H]DG than healthy cells.

Conclusions

These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

Activation of receptors negatively coupled to adenylate cyclase is required for induction of long‐term synaptic depression at Schaffer collateral‐CA1 synapses

Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral‐CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxy‐cyclopropyl) glycine (DCGIV; 5 μM), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 μM), resulted in a long‐lasting depression of synaptic strength. When zaprinast (20 μM) was combined with a cell‐permeant PKA inhibitor H‐89 (10 μM), the need for mGluR IIs was bypassed. DCGIV, when combined with a “submaximal” low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)‐alpha‐ethylglutamic acid (EGLU; 5 μM), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)‐a‐Cyclopropyl‐[3‐³H]‐4‐phosphonophenylglycine (CPPG; 10 μM), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N⁶‐cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 μM), was sufficient to elicit CLTD. Inhibition of PKA with H‐89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Treatment of nitric oxide supplemented with nitrogen and sulfur regulates photosynthetic performance and stomatal behavior in mustard under salt stress

Nitric oxide (NO) is a hormone that connects numerous reactions in plant cells under normal and environmental stress conditions. The application of 100 µM NO as sodium nitroprusside (SNP; NO donor) applied individually or in combination with N or S in different combinations (i.e. 100 mg N or S kg⁻¹ soil applied at the time of sowing [100 N + 100S]_0d or with split, 50 mg N or S kg⁻¹ soil at the time of sowing and similar dose at 20 d after sowing [50 N + 50S]_0d + [50 N + 50S]_20d) was tested to alleviate salt stress in mustard (Brassica juncea L.). Application of 100 µM NO plus split application of N and S more significantly promoted stomatal behavior, photosynthetic and growth performance in the absence of salt stress and maximally alleviated effects of salt stress through increased N- and S-use efficiency, proline and antioxidant system. The combined application of N and S at the time of sowing was lesser effective in promoting photosynthesis and growth under salt or no salt stress conditions in presence or absence of NO. The study suggests that salt stress effects on the photosynthetic performance are mitigated more efficiently when NO was applied together with the split application of N and S given at two stages, and the photosynthetic activity was promoted under salt stress through increased N and S assimilation and antioxidant system. This strategy may be adopted in agricultural system for overcoming salt stress effects on performance of mustard.     

Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP

Ca²⁺ changes induced by nitric oxide (NO^·) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1–100 μmol/L) and S-Nitroso-N-acetylpenicillamine (SNAP) (100 μmol/L) were used as NO^· donors. The cytoplasmatic Ca²⁺ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO^· donors caused transient oscillatory Ca²⁺ changes, which were not detectable in the presence of oxyhemoglobin (50 μmol/L). Digital ratio imaging revealed initiation sites within cells where Ca²⁺ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca²⁺-free buffer. L-type Ca²⁺-channel blocker diltiazem (100 μmol/L) was not able to block these responses. NO^·-induced Ca²⁺ release from intracellular stores caused capacitative Ca²⁺ entry. Both thapsigargin (1 μmol/L) and cyclopiazonic acid (10 μmol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 μmol/L) nor dantrolene (100 μmol/L) was able to inhibit Ca²⁺ changes induced by SNP, indicating that primarily inositol 1,4,5-triphosphate (IP₃)-dependent stores are released upon stimulation with NO^·. A small inhibitory effect of ATP- and SNP-induced peak [Ca²⁺]_i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO^·-induced Ca²⁺ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY (83583) nor cell permeant analogues of cGMP altered or simulated [Ca²⁺]_i changes. An inhibitor of cGMP-dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP₃ receptors to induce Ca²⁺ changes in endothelial cells stimulated with NO^·. J. Cell. Physiol. 172:296–305, 1997. © 1997 Wiley-Liss, Inc.     

A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. II. Effects on uptake and efflux of IAA in maize roots

An explanation is sought for the inhibition of maize root growth and gravireaction brought about by treatment with 3,5-diiodo-4-hydroxybenzoic acid (DIHB). The effects of DIHB and 2,3,5-triiodobenzoic acid (TIBA) on the uptake and efflux of [³H]-indol-3yl-acetic acid (IAA) were tested using segments prepared from the elongation zone (2 to 7 mm region) of maize (Zea mays L. cv. LG11) roots. The uptake of [³H]-IAA (21 nM) by root segments incubated in buffered solutions (pH 5.0) was measured over a 5-min time-course. No significant effect of DIHB at 100 μM was observed, whereas TIBA at 10 μM slightly stimulated the uptake of [³H]-IAA. This experiment was repeated with the addition of non-radioactive IAA (total IAA concentration 1.0 μM). Up to 3 min DIHB (100 μM) had no significant effect, but thereafter a slight stimulation of IAA net uptake was observed. Treatment with TIBA (10 μM) stimulated the accumulation of IAA in the segments. The effects of DIHB (10, 50, 100 μM) and TIBA (10 and 50 μM) on the efflux of [³H]-IAA from segments that had been pretreated in [³H]-IAA (22 nM) were then tested. Treatment with DIHB or TIBA at pH 5.0 inhibited IAA efflux; the inhibition by TIBA was more marked than that produced by DIHB. This experiment was repeated using DIHB (10, 50, 100 μM) buffered at pH 6.0, and an inhibition of IAA efflux was again observed. Both DIHB (10 μM) and TIBA (10 μM) inhibited the binding of [³H]-NPA to a 5000–48000 g membrane fraction prepared from whole maize roots. The effects of the two substances were similar: 40% inhibition of specific binding by DIHB and 41% inhibition by TIBA. This indicates that DIHB, like TIBA, binds to the N-1-naphthyl-phthalamic acid-sensitive carrier for IAA efflux. It is concluded that DIHB, like TIBA, inhibits IAA transport at the level of efflux. The similarity between DIHB and TIBA as regards chemical structure and their inhibitory effects on IAA efflux and NPA binding strongly suggest that they act on the same carrier for IAA efflux across the plasmalemma.     

Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.