Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus.
2. In physiological salt solution containing 4×10⁻⁶ M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5–32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5–4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8–32 Hz.
3. In the presence of L-N^G-nitroarginine methyl ester (L-NAME; 3×10⁻⁴ M) or physostigmine (10⁻⁶ M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone.
4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies.
5. Off-relaxations in the presence of L-NAME were partially reduced by α-chymotrypsin (10 U ml⁻¹).
6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME.
7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10⁻⁵ M), depending upon the degree of relaxation by SNP.
8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

     

Role of nitric oxide produced by constitutive and inducible nitric oxide synthases in the mouse gastric fundus

In the present study, the role of nitric oxide (NO) produced by constitutive and inducible nitric oxide synthases (cNOS and iNOS, resepctively) on the contraction and relaxation of fundus in normal and lipopolysaccharide (LPS)-treated mice was examined. A whole fundic ring isolated from mice pretreated with reserpine was mounted in an organ bath containing Krebs' solution with 0.001 mmol/L atropine. Rings were contracted initially by 5-hydroxytryptamine (5-HT; 0.03 mmol/L) before relaxation was induced using ATP (0.03 mmol/L), ADP (0.03 mmol/L), pentoxifylline (0.002 mmol/L), electrical field stimulation (EFS; 50 V, 1 msec, 50 Hz, 3 min) and L-arginine (0.05 mmol/L). All drugs and EFS induced significant relaxation of isolated rings. The relaxations induced were significantly inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; 1.0 mmol/L). However, the iNOS inhibitors L-N(6)-(1-iminoethyl) lysine hydrochloride (L-NIL; 1.0 mmol/L) and amino guanidine (AMG; 1.0 mmol/L) had no significant effect on tissue relaxation. Then, the relaxant effects of 0.03 mmol/L ATP were tested on precontracted isolated fundic rings taken from 10 mg/kg LPS-treated animals. The non-selective NOS inhibitor L-NAME (10 mg/kg), the iNOS inhibitors L-NIL (3 mg/kg) and AMG (20 mg/kg) and betamethasone (0.1 mg/kg) were used to examine the role of NO produced by iNOS in the relaxation responses. It was found that the level of contraction induced by 0.03 mmol/L 5-HT in rings isolated from LPS-treated animals was significantly (P < 0.5) less than that in rings from untreated mice. However, precontracted tissues from LPS-treated mice were significantly relaxed by ATP and the relaxation response to ATP was significantly inhibited by L-NIL, ANG and betamethasone, but not by L-NAME. We suggest that, in LPS-treated mice, the production of NO from iNOS produces a reduction in the contractile response, as well as a decrease in NO formation by cNOS, resulting in changes to smooth muscle cell function.     

Involvement of vasoactive intestinal polypeptide in nicotine-induced relaxation of the rat gastric fundus

1. Nicotine-induced relaxation and release of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus.
2. Under non-cholinergic conditions (0.3 μM atropine), nicotine (3–300 μM) produced concentration-dependent relaxations of the 5-hydroxytryptamine (3 μM)-precontracted strips. Under non-adrenergic non-cholinergic (NANC) conditions (0.3 μM atropine+1 μM phentolamine+1 μM nadolol), relaxations induced by sub-maximal nicotine concentrations (10 and 30 μM) were significantly smaller, while that produced by the highest concentration used (300 μM) was similar to that seen under non-cholinergic conditions.
3. Re-exposure to the same nicotine concentration 1 h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used.
4. Under non-cholinergic conditions, the relaxant response to 30 μM nicotine was abolished by hexamethonium (100 μM) and significantly reduced by tetrodotoxin (TTX, 3 μM). The TTX-resistant component was not observed under NANC conditions.
5. NANC relaxation induced by 30 μM nicotine was significantly reduced by a specific anti-VIP serum (approximately 35% less than that seen with normal rabbit serum).
6. Nicotine (30–300 μM) caused significant, concentration-dependent increases in the outflow of VIP- and PHI-LI from the strips; these effects were also diminished with re-exposure. The increases in both types of immunoreactivity evoked by nicotine (300 μM) were abolished by hexamethonium (300 μM), TTX (3 μM) and a calcium-free medium.
7. These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.

     

Influence of a selective guanylate cyclase inhibitor,and of the contraction level,on nitrergic relaxations in the gastric fundus

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus.
2. ODQ (10⁻⁶ and 10⁻⁵ M) concentration-dependently inhibited the short-lasting relaxations by NO (2×10⁻⁶ M–10⁻⁴ M) administered as a bolus without influencing the relaxation by 3×10⁻⁸ M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10⁻⁶ and 10⁻⁵ M ODQ.
3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5–16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10⁻⁶ and 10⁻⁵ M ODQ, and by the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 3×10⁻⁴ M).
4. ODQ (10⁻⁶ M) and L-NAME (3×10⁻⁴ M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz).
5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F_2α (PGF_2α), the inhibitory effect of 3×10⁻⁴ M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3×10⁻⁷ M PGF_2α.
6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.

     

Influence of polyethylene-glycol-superoxide dismutase and combined depletion and repletion of antioxidants on nitrergic relaxation in the pig gastric fundus

In circular smooth muscle strips of porcine gastric fundus, polyethylene-glycol-superoxide dismutase, a membrane-permeable analogue of endogenous copper/zinc (Cu/Zn) superoxide dismutase, reversed the inhibitory effect of the superoxide anion generator 6-anilino-5,8-quinolinedione (LY83583) on electrically induced nitrergic relaxations of fundic tissues which are depleted of the endogenous antioxidant Cu/Zn superoxide dismutase by diethyldithiocarbamate, to the same extent as exogenously added Cu/Zn superoxide dismutase. Addition of a second antioxidant together with Cu/Zn superoxide dismutase does not result in a higher degree of reversal of the inhibitory effect of LY83583. Depletion of either tissue glutathione or tissue catalase in combination with diethyldithiocarbamate does not increase the inhibitory action of LY83583 or the nitric oxide (NO)-scavenger hydroxocobalamin upon nitrergic relaxations (electrically induced or by exogenous NO) when compared to their action in the presence of diethyldithiocarbamate alone. In conclusion, these results demonstrate that endogenous Cu/Zn superoxide dismutase is the essential antioxidant responsible for safeguarding peripheral nitrergic neurotransmission, whereby extracellular protection of endogenous NO is most important.     

Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta

Background and purpose:

Nebivolol is a highly selective β₁-adrenoceptor antagonist with β₃-adrenoceptor agonist properties and is a racemate mixture of D- and L-enantiomers. However, the cellular mechanisms of the effects of each enantiomer are not yet clear and are a matter for debate. The aim of the present experiments was to determine the adrenoceptors involved in the vascular effects of D- and L-enantiomers of nebivolol in rat thoracic aorta.

Experimental approach:

Responses to nebivolol enantiomers were evaluated in rings of thoracic aorta from male Sprague-Dawley rats.

Key results:

D-nebivolol (0.1–10 µmol·L⁻¹), but not L-nebivolol, significantly shifted to the right the concentration-response curve to phenylephrine, an α₁-adrenoceptor agonist, in a concentration-dependent manner. For the following experiments, aortic rings were constricted with endothelin 1 and now both enantiomers produced an endothelium-dependent relaxation of the rings involving the nitric oxide pathway. This relaxation was not modified by 1 µmol·L⁻¹ CGP 20,712A (β₁-adrenoceptor antagonist), but significantly blunted by 7 µmol·L⁻¹ L-74,8337 (β₃-adrenoceptor antagonist). However, only the vasorelaxation induced by D-nebivolol was significantly reduced by 1 µmol·L⁻¹ ICI 118,551 (β₂-adrenoceptor antagonist).

Conclusions and implications:

Our results suggest that the nebivolol enantiomers act on different targets. D-nebivolol induced vasorelaxation by activating β₂- and β₃-adrenoceptors and antagonizing α₁-adrenoceptors. L-nebivolol induced vasorelaxation by activating only β₃-adrenoceptors in our model. Our results emphasize that nebivolol is a β₁-adrenoceptor antagonist with several important pharmacological differences from other β₁-adrenoceptor antagonists.     

The adrenergic,cholinergic and NANC nerve-mediated contractions of the female rabbit bladder neck and proximal,medial and distal urethra

1. The nerve-mediated contraction of the female rabbit bladder neck and different portions of the urethra (proximal, medial and distal) was studied in vitro by electrical stimulation (50 V, 30 Hz, 0.05 ms width, trains of 5 s every 5 min) by use of a superfusion system.
2. The amplitude (E_max) and the duration (D_max) of the stimulated contraction were studied in the four tissues. The E_max value was significantly higher in distal urethra (2.07±0.15 g) compared to the bladder neck (1.08±0.10 g), proximal urethra (0.73±0.07 g) and medial urethra (0.87±0.07 g). In contrast, the D_max value appeared slightly but significantly lower (P<0.05) in distal urethra (68.5±2.3 s) than in bladder neck (76.7±6.0 s), proximal urethra (84.5±5.0 s) and medial urethra (81.3±3.5 s).
3. Cocaine (1 μM) significantly increased the basal E_max values in medial and distal urethra and the basal D_max values in the four tissues.
4. Prazosin (1 μM) significantly reduced E_max value in proximal, medial and distal urethra and D_max value in bladder neck and proximal urethra. Atropine (1 μM) also significantly reduced E_max values in bladder neck and proximal urethra and reduced D_max value in bladder neck, but not in other tissues. Yohimbine (0.1 μM) was devoid of effect in the four tissues.
5. The association of prazosin (1 μM) and atropine (1 μM) did not modify the E_max and the D_max values of the electrically-induced contractions, except in proximal urethra and in bladder neck where an additive inhibitory effect (on E_max only) was observed compared to prazosin and atropine alone.
6. The residual contractile response after combined treatment with prazosin and atropine was significantly diminished by tetrodotoxin (TTX; 1 μM) but not completely abolished. These NANC contractions were insensitive to P2X-purinoceptor desensitization by continuous tissue perfusion with α,β-methylene ATP (30 μM).
7. These results demonstrate that bladder neck and proximal urethra are mainly innervated by the parasympathetic nervous system, whereas medial and distal urethras are to a greater extent under the control of the sympathetic innervation. The residual responses, insensitive to prazosin and atropine, may indicate a NANC innervation in the four tissues. However, the nature of the NANC neurotransmitter remains to be identified.

     

Effect of 3-isobutyl-1-methylxanthine and zaprinast on non-adrenergic non-cholinergic relaxation in the rat gastric fundus

Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) have been proposed as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in the rat gastric fundus. The smooth muscle relaxant actions of VIP and NO are mediated by cAMP and cGMP, respectively; therefore the effect of inhibitors of phosphodiesterases responsible for cyclic nucleotide breakdown on relaxation induced by VIP, NO and electrical field stimulation was investigated. The non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), the cGMP-specific phosphodiesterase inhibitor, zaprinast, the adenylate cyclase activator, forskolin, and the cyclic nucleotide analog, 8-bromo cGMP, produced concentration-dependent relaxation of rat gastric fundus strips precontracted by PGF₂. IBMX potentiated isoprenaline-induced relaxation but not relaxation induced by sodium nitroprusside, VIP, NO or electrical field stimulation. Zaprinast potentiated the relaxation induced by sodium nitroprusside, while having no influence on relaxation due to any other stimulus. The combination of both phosphodiesterase inhibitors did not significantly affect the electrically induced relaxation. It is concluded that both cAMP and cGMP mediate relaxation in the rat gastric fundus. Further research is needed to investigate the role of the cyclic nucleotides during NANC relaxation of this tissue.     

Influence of thiorphan and phosphoramidon on the relaxant effect of vasoactive intestinal polypeptide and non-adrenergac non-cholinergic neurone stimulation in the rat gastric fundus

Relaxations were induced in longitudinal muscle strips of the rat gastric fundus by exogenous administration of vasoactive intestinal polypeptide (VIP) and by transmural stimulation in the presence of atropine. These responses were not influenced by two neutral endopeptidase inhibitors, thiorphan (3 × 10⁻⁵ M) and phosphoramidon (10⁻⁵ M). This suggests that neutral endopeptidase is not involved in the breakdown of exogenous VIP and non-adrenergic non-cholinergic inhibitory transmitters of the rat gastric fundus.     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

1. We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.
2. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48±5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production.
3. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10  mM) to potentiate LPS-induced NO synthesis.
4. Metabolism of L-[³H]arginine to L-[³H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mM) or by putrescine (0.001–1 mM), spermidine (0.001–1 mM) or spermine (0.001–1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L-NMMA (100 μM).
5. Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.
6. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

     

Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages

1. The ability of three modified tetrapeptides, representing fragments of the C-reactive protein (CRP) sequence and stabilized in the first peptide bond by retro-inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice.
2. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP I, H-gThr-(R,S)mLys-Pro-Leu-OH, ITF 1192; CRP II, H-gGly-(R, S)mLys-Pro-Arg-OH, ITF 1127; CRP III, H-gThr-(R,S)mLys-Pro-Gln-OH, ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose-dependent manner; the most stimulating dose was 1000 ng ml⁻¹ for CRP II and 100 ng ml⁻¹ for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation.
3. The enhanced effect of retro-inverso CRP-related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones.
4. The production of NO by retro-inverso CRP-peptide analogues was significantly inhibited by dexamethasone (20 μM), N^G-monomethyl-L-arginine (L-NMMA) (500 μM) and pyrrolidine dithiocarbamate (PDTC) (100 μM).
5. Retro-inverso CRP-peptide analogues stimulated macrophages to produce high levels of interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α) in the presence of LPS.
6. Retro-inverso CRP-peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL-1 and TNF-α, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti-IL-1 and anti-TNF monoclonal antibodies (mAbs) reduced retro-inverso analogue-induced NO secretion. Data indicate a predominant role for IL-1α in the induction of NO secretion by retro-inverso analogues.
7. These results suggest that retro-inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor-κB (NF-κB).

     

Mechanism of Curcuma longa and Its Neuroactive Components for the Management of Epileptic Seizures: A Systematic Review

Curcuma longa (Turmeric) is a tropical herbaceous perennial plant of the family Zingiberaceae and contains curcuminoids, sesquiterpenoids and monoterpenoids as its major components. Given the broad range of activities that Curcuma longa possesses and also its use as a traditional epilepsy remedy, this review attempts to systematically review the experimentally proven activities of Curcuma longa and its bioactive components, which are related to the management of epileptic seizures. Using the PRISMA model, five databases (Google Scholar, PubMed, ScienceDirect, SCOPUS and SpringerLink) were searched using the keywords [“Curcuma longa” AND “Epilepsy”] and [“Curcuma longa” AND “Seizures”], leaving 34 articles that met the inclusion criteria. The present systematic review elaborated on the experimentally proven potential of Curcuma longa components, such as an aqueous extract of Curcuma longa itself, Curcuma longa oil and active constituents like curcuminoids and bisabolene sesquiterpenoids found in Curcuma longa with anti-seizure potential. Using human equivalent dose calculations, human treatment parameters were suggested for each component by analysing the various studies in this review. This review also determined that the principal components possibly exert their anti-seizure effect via the reduction of corticosterone, modulation of neurotransmitters signalling, modulation of sodium ion channels, reduction of oxidative DNA damage, reduction of lipid peroxidation, upgregulation of brain-derived neurotrophic factor (BDNF) and γ-aminobutyric acid (GABA) mediated inhibition. It is anticipated that this review will help pave the way for future research into the development of Curcuma longa and its neuroactive constituents as potential drug candidates for the management of epilepsy.     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and α-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α-CGRP were also investigated.
2. Adrenomedullin and α-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC₅₀ values for adrenomedullin and α-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, N^G-monomethyl-L-arginine (L-NMMA, 100 μM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 μM).
3. Adrenomedullin and α-CGRP increased the endothelial [Ca²⁺]_i in the rat aorta with endothelium, whereas they did not change [Ca²⁺]_i in the smooth muscle.
4. An antagonist of the CGRP₁ receptor, CGRP (8–37), antagonized the relaxant effects of α-CGRP and the β-isoform of CGRP (β-CGRP) but not those of adrenomedullin in the rat aorta.
5. In the porcine coronary artery contracted with U46619, adrenomedullin and α-CGRP caused a concentration-dependent relaxation with an IC₅₀ of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC₅₀ values nor the maximal relaxations induced by adrenomedullin or α-CGRP. When the artery was contracted with high K⁺ solution (72.7 mM), these peptides caused a small relaxation.
6. Adrenomedullin and α-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca²⁺]_i in the porcine coronary artery.
7. CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β-isoform of CGRP (β-CGRP).
8. These results suggest that in the rat aorta, adrenomedullin and α-CGRP increase the endothelial [Ca²⁺]_i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca²⁺]_i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α-CGRP, β-CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α-CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β-CGRP.

     

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose:

Recent findings suggest that the noxious gas H₂S is produced endogenously, and that physiological concentrations of H₂S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H₂S to modulate carrageenan-induced synovitis in the rat knee.

Experimental approach:

Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H₂S formation (dl-propargylglycine) or an H₂S donor [Lawesson''s reagent (LR)].

Key results:

Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters.

Conclusions and implications:

Whereas exogenous H₂S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H₂S exerts little immunomodulatory effect. These data further support the development and use of H₂S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.     

Rapid characterization of metabolites in soybean using ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) and screening for α-glucosidase inhibitory and antioxidant properties through different solvent systems

This work was the first to investigate on the simultaneous characterization of metabolite profiles in soybean using UPLC-ESI-Q-TOF-MS/MS. Twenty two compositions were observed within 14 min from the methanol extract and confirmed as twelve isoflavones of three types and ten soyasaponins (Ab, Af, I–III, αg, βg, βa, γg, and γa). Moreover, the patterns of two chemicals showed considerable differences in seven solvent systems by HPLC analysis and their optimal extraction was achieved by 70% methanol (isoflavone: 4102.69 μg/g; soyasaponin: ten peaks). The second abundant isoflavones were detected in 50% methanol (4054.39 μg/g), followed by 30% methanol, 100% methanol, 10% methanol, CH₂Cl₂, and acetone extracts with 3134.03, 2979.49, 1681.33, 366.19, and 119.00 μg/g, respectively. Soyasaponins exhibited similar tendencies as those of isoflavones. The highest total phenolic was found as 2.10 ± 0.05 mg GAE/g in 70% methanol with remarkable differences by comparing other extracts. Specifically, this extract showed potent α-glucosidase inhibitory (81%) and antioxidant capacities (DPPH: 93% and ABTS: 95%) at a concentration of 1.0 mg/mL. Our results may be contributed to enhancing the value to functional foods and evaluating the secondary metabolites concern to antioxidant properties using solvent system in soybean.     

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection.
2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7±0.9 nmol g⁻¹ tissue). This release was increased by 96% when muscles were incubated in high K⁺ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 μM), ω-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 μM), aspartate (100 μM), γ-aminobutyric acid (100 μM) or carbachol (50 μM).
3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6±12.7 nmol g⁻¹ tissue but, unlike ascorbate, urate release was unchanged in high K⁺ PSS. In functional experiments, urate (100–400 μM) partially protected NO (15 μM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 μM), but not from inhibition by hydroquinone or duroquinone (both 100 μM).
4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 μM; 2 h) still exhibited release of ascorbate (2.5±0.4 nmol g⁻¹ tissue) and urate (22.2±2.9 nmol g⁻¹ tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K⁺ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles.
5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

     

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action

Background and purpose

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.

Experimental approach

JZL184 and/or the CB₁ receptor antagonist, AM251 or the CB₂receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.

Key results

JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE₂ and PGD₂ were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.

Conclusion and implications

Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Differential effect of L-NAME and S-methyl-isothiourea on leukocyte emigration in carrageenin-soaked sponge implants in rat

1. The role of nitric oxide (NO) in leukocyte (polymorphonuclear cells, monocytes and lymphocytes) emigration was studied in a model of carrageenin-sponge implants in rats.
2. The subcutaneous implantation of 1% (w/v) of λ-carrageenin-soaked sponges elicited an inflammatory response that was characterized by a time-related increase in leukocyte infiltration in the sponges and increased levels of nitrite in the exudate. Total leukocyte infiltration and nitrite production were maximal at 24 h and decreased after 48 and 96 h. The mononuclear cell influx was maximal at 48 h (21% of the total leukocytes). Therefore, this time point was used in the successive experiments.
3. Polymorphonuclear cell (PMN) and lymphocyte infiltration in the sponges significantly increased when rats were treated with the non-specific NO-synthase (NOS) inhibitor, N^G-nitro-L-arginine methylester (L-NAME) (1 mg ml⁻¹ in drinking water ad libitum). Monocyte emigration was not affected by L-NAME treatment. The nitrite levels in the exudate of L-NAME-treated rats were significantly reduced. The concomitant ingestion of L-arginine (30 mg ml⁻¹) resulted in a reversion of the L-NAME effect, while D-arginine (30 mg ml⁻¹) had no effect, indicating the involvement of the L-arginine: NO pathway.
4. Administration of L-NAME resulted also in an increased release of tumour necrosis factor-α (TNF-α) and prostacyclin (measured as the stable metabolite, 6-keto-PGF_1α). L-NAME had no effect on monocyte chemoattractant protein-1 (MCP-1) release in the exudate.
5. Since L-NAME may have effects on the local blood flow, phenylephrine (0.034 mg ml⁻² in drinking water) was used as it has an effect on the local blood flow similar to L-NAME. Phenylephrine had no effect on either leukocyte emigration, or on nitrite, TNF-α, prostacyclin or MCP-1 accumulation in the exudate.
6. In contrast, the more selective iNOS inhibitor S-methyl-isothiourea (SMT) (10 μg ml⁻¹ in drinking water) significantly reduced PMNs and lymphocyte influx in the sponge, having no effect on monocyte influx. Moreover, SMT decreased nitrite production in the exudate to a comparable extent as L-NAME.
7. Administration of SMT significantly reduced MCP-1 release in the exudate, without an effect on TNF-α or prostacyclin production. Moreover SMT did not produce any changes in local blood flow.
8. Our results show that a different outcome of the inflammatory process can be obtained depending on the types of NOS inhibitor used.

     

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures.
2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 μM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors.
3. Daily treatment with nitric oxide synthase inhibitors, such as N^G-nitro-L-arginine methyl ester (L-NAME, 300 μM), led to the stimulation of cell proliferation by 44±5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth.
4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect.
5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-γ and uncoupling protein-1, which are upregulated during differentiation.
6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100–1000 μM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-γ and uncoupling protein-1.

     

Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells

1. In RAW 264.7 murine macrophages and rat aortic smooth muscle (RASM) cells lipopolysaccharide (LPS) alone or in combination with interferon γ (IFNγ) or forskolin, respectively, stimulated the expression of the 130 kDa inducible isoform of nitric oxide synthase (iNOS) in both a time- and concentration-dependent manner.
2. Incubation with the direct activator of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) alone, did not result in detectable iNOS expression in either cell type.
3. Chronic PMA pretreatment resulted in significant down-regulation of α, β and ε isoforms of PKC in RAW 264.7 macrophages and corresponded to a 20–30% reduction in LPS-induced iNOS expression. In contrast, IFNγ alone or in combination with LPS stimulated an approximate 20% and 50% potentiation, respectively.
4. Pre-incubation with PKC inhibitors (calphostin C and H-7) showed similar effects upon stimulated induction of iNOS.
5. In RASM cells chronic PMA pretreatment resulted in down-regulation of α and ε PKC isoforms and corresponded to potentiation of iNOS expression in response to LPS alone or in combination with forskolin.
6. Co-incubation of RASM cells in the presence of PMA, angiotensin II (AII) or foetal calf serum (FCS) resulted in the inhibition of iNOS expression in response to LPS alone or in combination with forskolin.
7. Differential sensitivity to PKC inhibitors (calphostin C and H-7) was observed in RASM cells and exhibited both negative and positive modulation of stimulated induction.
8. In addition the PKC inhibitor compound Ro-31-8220 abolished stimulated induction in both cell types in response to all treatments.
9. These results suggest that PKC activation is required for induction of the 130 kDa isoform of NOS in both RAW 264.7 macrophages and RASM cells. However, individual PKC isoforms regulate iNOS expression in both a positive and negative manner.