S. Franchi-Micheli

Agonists, antagonists and modulators of excitatory amino acid receptors in the guinea‐pig myenteric plexus

1 The receptors for glutamic acid (L‐Glu) present in the guinea‐pig myenteric plexus‐ileal longitudinal muscle preparation have been studied by measuring the muscle contraction induced by numerous putative endogenous agonists acting at these receptors. Furthermore, the actions of different concentrations of antagonists, glycine, Mg2+ and Ca2+ on the ileal contractions induced by l‐G1u have been evaluated. 2 The EC50 values of the most common putative endogenous agonists of these receptors were: L‐Glu 1.9 × 10−5 m; L‐aspartate 8 × 10−5 m; quinolinate 5 × 10−4M; L‐homocysteate 1.4 × 10−4M; the dipeptide aspartyl‐glutamate 8 × 10−5m, while N‐acetyl‐aspartyl‐glutamate was inactive. Among the molecules used to classify excitatory amino acid receptors, N‐methyl‐D‐aspartate (NMDA) was the most potent (EC50 5 × 10−4m). Kainic and quisqualic acids were almost completely inactive. 3 The responses to L‐Glu were competitively antagonized by 2‐amino‐5‐phosphonovaleric acid. They were, also, prevented by hyoscine (10−7m) and by tetrodotoxin (3 × 10−7 m), suggesting that the L‐Glu‐induced ileal contraction was in some way dependent upon an action on the myenteric cholinergic neurones. Kynurenic acid was a non‐competitive antagonist, γ‐D‐glutamyl‐taurine (10−4m) and aminophosphonobutyric acid (10−4m) did not modify the L‐Glu‐induced contractions. 4 Glycine (10−5 m) significantly potentiated the effects of glutamate especially when the ionic composition of the superfusion medium contained concentrations of Ca2+ in the range of 0.6–1.2 mM. Strychnine 3 × 10−5 m did not modify the actions of glycine. 5 The data presented here confirm the presence of NMDA receptors in the guinea‐pig myenteric plexus, and show that these receptors, similar to those present in primary neuronal cultures may be modulated by glycine.

The ACh‐induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells

Our previously published data indicate that an endogenously produced 5‐lipoxygenase metabolite can strongly contract isolated endothelium‐preserved rat aortic strips when cyclo‐oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl‐containing leukotrienes (Cys Lts) are involved in this endothelium‐dependent contraction. The isometric contraction of endothelium‐preserved rat aortic strips was recorded in preparations preincubated with 5 μM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK‐571, selective type 1 Cys Lts receptor (Cys Lt1) antagonists and the Cys Lt1/Cys Lt2 (type 2 Cys Lts receptor) antagonist BAYu9773 dose‐dependently prevented ACh‐induced contraction, their IC50s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC50 1.5 μM). In rat aorta smooth muscle cells (RASMs), Western blot analysis showed the presence of Cys Lt1 and Cys Lt2 receptors, the Cys Lt1 receptor being predominantly expressed. In fura‐2 loaded RASMs, LTD4 (0.01–100 nM) and LTC4 (200–800 nM) dose‐dependently increased intracellular calcium concentration ([Ca2+]i). Montelukast (1–100 nM) reduced LTD4‐induced [Ca2+]i increase, its IC50 being approximately 10 nM. BAY u9773 exhibited significantly low effectiveness. LTD4 (10 nM) induced a redistribution of smooth muscle actin fibres throughout the cytoplasm as visualized by confocal microscopy. In conclusion, Cys Lt1 activation by endogenously produced Cys Lts, can contract rat aortas, while Cys Lt2 only marginally influences aortic tone. Intracellularly, this effect is mediated by an increase in [Ca2+]i. Therefore, Cys Lts, by inducing vascular contraction, can contribute to systemic hypertension.

Lipid peroxidation induced “in vivo” by iron-carbohydrate complex in the rat brain cortex

In view of the emerging role of metals and particularly iron in the pathogenesis of several ischemic or degenerative CNS diseases, via a lipid peroxidative process, a model of slow iron-induced peroxidative damage in the rat brain cortex has been carried out. Iron-carbohydrate complexes were injected in the right brain cortex, and biochemical assays were performed on ipsilateral and contralateral samples two hours or seven days after injection. Iron-sacchararate caused a significant increase in the ipsilateral cortex in TBARS, conjugated dienes and fluorescent substances seven days after injection, whereas no biochemical alteration was observed two hours after treatment. In order to prevent or to limit lipid peroxidation, some drugs known for chelating and/or scavening activity were administered to iron-injected rats. DL-α-tocopherol, methylprednisolone, D-penicillamine significantly decreased the value of fluorescent products formed by iron-saccharate, whereas desferrioxamine was not effective.

GABAB receptor-mediated mechanisms in human intestine in vitro

The spontaneous motility of longitudinal muscle of human jejunum was recorded and the effect of gamma-aminobutyric acid-ergic (GABAergic) drugs was tested. GABA and (-)-baclofen (10(-6)-10(-4) M) dose dependently reduced the amplitude and frequency of the spontaneous contractions; muscimol and 3-aminopropanesulfonic acid (3 x 10(-5) M) were ineffective. The effect of 3 x 10(-5) M GABA was reduced by 3 x 10(-3) M 5-aminovaleric acid but not by 3 x 10(-5) M picrotoxin. The dose-response curve for GABA was shifted to the right by 3 x 10(-3) M 3-aminopropanesulfonic acid. Tetrodotoxin 3 x 10(-7) M prevented the GABAergic action, whereas various receptor antagonists tested did not affect it. GABAergic drugs did not influence the spontaneous motility of either circular or longitudinal muscles of human colon. It is suggested that GABAB receptor activation induces the inhibition of human jejunum longitudinal muscle motility by a neurogenic mechanism. The possible involvement of postganglionic cholinergic neurons is to be evaluated by other techniques.

Cyclooxygenase activity of hemoglobin

Publisher Summary When hemoglobin (Hb) diffuses into the extracellular space of living tissues, which may occur during hemolysis or when erythrocytes escape from vessels, its catalytic potential toward several endogenous or exogenous compounds can be activated. There has been much attention focused on the capacity of Hb to metabolize polyunsaturated fatty acids (PUFAs), giving rise to the formation of products that may be important in the pathogenesis of systemic (intravascular hemolysis) or local (micro- or macrohemorrhages, inflammation, hemorrhagic infarcts) conditions in which erythrocyte lysis occurs. At low concentrations, Hb catalyzes a quasi-lipoxygenase reaction on linoleic acid with remarkably high substrate specificity. The fatty acid cyclooxygenases, which may be regarded as a special type of lipoxygenase that initiate the biosynthesis of prostaglandins and thromboxanes, require heme for their activity. Hb is a good candidate for exerting cyclooxygenase activity owing to its peroxidative properties already described in the chapter. There is also some discussion about the cyclooxygenase activity of Hb, which has been made possible by the use of either physicochemical or biological analytical method.

Losartan counteracts the hyper-reactivity to angiotensin II and ROCK1 over-activation in aortas isolated from streptozotocin-injected diabetic rats

BackgroundIn streptozotocin-injected rats (STZ-rats), we previously demonstrated a role for angiotensin II (AT-II) in cardiac remodelling and insulin resistance partially counteracted by in vivo treatment with losartan, an AT-II receptor antagonist.We now aimed to investigate the effect of treating diabetic STZ-rats with losartan on diabetes vascular response to vasoconstrictors.MethodsMale Wistar rats were randomly divided in four groups, two of them were assigned to receive losartan in the drinking water (20 mg/kg/day) until the experiment ending (3 weeks afterward). After 1 week, two groups, one of which receiving losartan, were injected in the tail vein with citrate buffer (normoglycemic, N and normoglycemic, losartan-treated, NL). The remaining received a single injection of streptozotocin (50 mg/kg in citrate i.v.) thus becoming diabetic (D) and diabetic losartan-treated (DL). Plasma glycaemia and blood pressure were measured in all animals before the sacrifice (15 days after diabetes induction).In aortic strips isolated from N, NL, D and DL rats we evaluated i) the isometric concentration-dependent contractile response to phenylephrine (Phe) and to AT-II; ii) the RhoA-kinase (ROCK1) activity and expression by enzyme-immunoassay and Western blot respectively.Key resultsThe concentration-dependent contractile effect of Phe was similar in aortas from all groups, whereas at all concentrations tested, AT-II contraction efficacy was 2 and half and 1 and half times higher in D and DL respectively in comparison with N and NL. AT-II contracture was similarly reduced in all groups by AT-II receptor antagonists, irbesartan or irbesartan plus PD123319. HA-1077 (10 μM), an inhibitor of ROCK1 activity, reduced AT-II efficacy (Δmg/mg tissue w.w.) by -3.5 ± 1.0, -4.6 ± 1.9, -22.1 ± 2.2 and -11.4 ± 1.3 in N, NL, D and DL respectively). ROCK1 activity and expression were higher in D than in N/NL and DL aortas.Conclusion and implicationsAortas isolated from STZ-rats present hyper-contracture to AT-II mainly dependent on the up-regulation of ROCK1 expression/activity. In vivo losartan treatment partially corrects AT-II hyper-contracture, limiting the increase in ROCK1 expression/activity. These data offer a new molecular mechanism supporting the rationale for using losartan in the prevention of diabetic vascular complications.

Effect of baclofen on different models of bronchial hyperreactivity in the guinea-pig

In this paper we report an inhibitory effect of (−)-baclofen on many models of bronchial hyperreactivity bothin vivo andin vitro. (−)-Baclofen protects guinea-pigs from the anaphylactic bronchospasm induced in sensitized animals by an ovalbumin aerosol and from that induced by aerosols of histamine and PGF2α. Moreover (−)-baclofen reduces the TXA2 and TXB2 output induced by ovalbumin from isolated sensitized guinea-pig lungs. On the other hand (−)-baclofen does not show antihistaminic, anticholinergic or antiprostaglandinic action on isolated tracheal preparations. It is concluded that baclofen can provide protection from bronchial hyperreactivity possibly through a modulation of autonomic nervous system activity.

Mechanical stretch reveals different components of endothelial‐mediated vascular tone in rat aortic strips

Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium‐preserved strips were precontracted with either phenylephrine or prostaglandin F2α (PGF2α). In control conditions, acetylcholine (ACh) dose‐dependently relaxed phenylephrine‐precontracted strips independently of resting tension. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh‐induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose‐response curve to the right. After preincubation with indomethacin (5 μM) or ibuprofen (10 and 100 μM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium‐dependent, dose‐dependent contraction. ACh (10−6 M) increased the contraction up to two times greater the phenylephrine‐induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF2α. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX‐derived metabolites, can relax precontracted strips. COX inhibition up‐regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase‐dependent, ACh‐induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh‐induced relaxation marginally.

D-penicillamine affects lipid peroxidation and iron content in the rat brain cortex

Abstractd-Penicillamine, a trifunctional aminoacid known for its ability to form metal complexes and for being a radical scavenger, has been investigated “in vitro” and “in vivo” in the rat brain cortex. At 50 μM the drug facilitate lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of thed-penicillamine (25 and 50 mg/Kg i.p) was evaluated “in vivo” after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated withd-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe2+/Fe3+ ratio was significantly different from that of normal rats. On the contrary ratios obtained formd-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest thatd-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agents, can remove metal from action sites where lipid peroxidation may occur.

Hemoglobin affects lipid peroxidation and prostaglandin E2 formation in rat corticocerebral tissues in vitro

Variations of lipid peroxidation and arachidonic acid (AA) metabolism products were found when experimental subarachnoid hemorrhage or ischemia and reperfusion were performed in an animal brain model. In a previous study, we showed that hemoglobin (Hb) produces prostaglandins when incubated in AA. To elucidate how Hb affects lipid peroxidation and AA metabolism in the CNS, we measured lipid hydroperoxides (LOOH), PGE2 and thiobarbituric acid reactant substances (TBARS) in corticocerebral homogenates and slices of rats (normal rats) after incubation with different concentrations (10(-9) to 10(-5) M) of Hb. In addition, brain cortices of indomethacin-treated (40 mg/Kg) rats (IN-treated rat) were incubated in the presence of 10(-5) M indomethacin (IN) to exclude the interference of prostaglandin enzyme synthetase. Hb was able to affect LOOH, PGE2, and TBARS production in both normal and IN-treated rat brain cortex homogenates and slices. In all cases, we found an increase in prostaglandin when 10(-8) M Hb was used, whereas no effect was noticed with 10(-9) M. On the other hand, with higher Hb concentrations (10(-6)-10(-5) M), the LOOH and PGE2 values did not reach statistical significance, and TBARS significantly increased. In all cases, when 10(-4) M scavenger or metal-chelating compounds were added to an incubation mixture with 10(-8) M Hb, PGE2 formation was inhibited, whereas no variation occurred when 10(-4) M IN was further added to IN-treated rat corticocerebral homogenate or slices. We hypothesize that in in vivo experimental neuropathologies, Hb must attain the 10(-8) M concentration in the reaction cellular microenvironment to stimulate PGE2 production, and that an evaluable part of this PGE2 production may be directly ascribable to the iron-heme oxy-redoxy activity of Hb.