Arachidonic acid products-mediated contraction induced by bradykinin in relaxed mesenteric arterial rings from Holtzman rats

This study describes the contractile action of bradykinin on rat isolated mesenteric arterial rings and a possible mechanism responsible for this action. Bradykinin induced dose-dependent contraction of relaxed mesenteric arterial rings from Holtzman rats, but not from Wistar rats. A second bradykinin challenge in the same ring induced a very small effect or no effect at all. Destruction of the endothelium did not modify the response to bradykinin. des-Arg⁹-[Leu⁸]bradykinin failed to antagonize bradykinin's action. HOE 140 (

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-Arg-[Hyp³,Thi⁵,

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-Tic⁷,Oic⁸]bradykinin) reduced bradykinin-induced contractions. Indomethacin abolished the contractile response to bradykinin; prostaglandin F₂ induced a long-lasting contraction, dissimilar from that induced by bradykinin; L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]-2,2-dimethyl propanoic acid), an antagonist of the thromboxane receptor, inhibited bradykinin-induced contractions. These results suggest that bradykinin-induced contraction in mesenteric arterial rings is indirect, through activation of bradykinin B₂ receptors, resulting in liberation of prostanoids from outside the endothelium. Thromboxane A₂ is probably an intermediate in this response but we cannot exclude the participation of other prostanoids.     

14β-Chlorocinnamoylamino derivatives of metopon: long-term μ-opioid receptor antagonists

The affinity, selectivity and antinociceptive properties of 5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [³H][

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-Ala²,(Me)-Phe⁴,Gly(ol)⁵]enkephalin (DAMGO) with IC₅₀ values of less than 2 nM. Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of μ-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of μ-opioid binding sites without a change in affinity. In the mouse 55°C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by δ- or κ-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, μ-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the μ-opioid receptor.     

Structure–activity relationships of astemizole derivatives for inhibition of store operated Ca channels and exocytosis

The effects of a series of analogues of the antiallergic drug astemizole on the exocytosis of the enzyme β-hexosaminidase were studied in a mast cell model, the rat basophilic leukemia (RBL-2H3) cell. Besides differences in the effects on FcεRI receptor-stimulated exocytosis, changes were also observed in Ca²⁺ influx and in the perturbation of the cell membrane. A strong correlation was found between the effects on antigen- and thapsigargin-stimulated

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influx. Furthermore, the inhibition of

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influx was correlated with the inhibition of β-hexosaminidase release and membrane stabilization. It is concluded that the astemizole analogues are capable of inhibiting mast cell β-hexosaminidase release through inhibition of Ca²⁺-store-operated Ca²⁺ channels (SOC). Compounds with high lipophilicity also released Ca²⁺ from intracellular stores. Lowering of the hydrophobicity by introduction of nitrogens or truncation at different sites in the astemizole structure decreased inhibitory activity on SOC channels. The inhibition of SOC channels cannot completely be ascribed to non-specific membrane effects. The piperidinyl–benzimidazole moiety was found to be important for inhibition of SOC channels. The observed differences in activity possibly depend on the way the compounds penetrate the membrane bilayer. Astemizole is an interesting new tool to study SOC channels and can be a lead for the design of mast cell-stabilizing antiallergic drugs.     

Anorectic action of bombesin requires receptor for corticotropin-releasing factor but not for oxytocin

The marked functional similarities between pharmacological effects of bombesin and of corticotropin-releasing factor (CRF), prompted the formulation and testing of our working hypothesis that BN may elicit its biological effects through the release of CRF. Central pretreatment with CRF receptor antagonists, -helical CRF-(9–41) (-CRF-(9–41)) or [

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-Phe¹², CMeLeu³⁷]CRF-(12–41) (CMeCRF), blocked the effects of centrally administered bombesin on food intake and related behaviors and partially attenuated the satiety effects of systemically administered bombesin. We also attempted to characterize the specificity of this interaction through the combined use of bombesin with the oxytocin antagonist, [d(CH₂)₅, Tyr(OMe)², Orn⁸]vasotocin (vasotocin). Central pretreatment with vasotocin failed to alter bombesin-induced behaviors, suggesting the absence of a pharmacological interaction between these two peptidergic systems. Finally, the CRF antagonist failed to reverse the oxytocin-induced suppression of food intake, indicating that CRF does not have a direct role in the mediation and/or modulation of the effects of oxytocin on food intake. Thus, the present experiments support the contention that bombesin partly mediates its feeding-suppressant effects through interactions with CRF. The specificity of this interaction is supported by the lack of interaction between bombesin and/or CRF with oxytocin.     

Inhibition of N-, P/Q- and other types of Ca channels in rat hippocampal nerve terminals by the adenosine A1 receptor

The effects of the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca²⁺]_i stimulated with 4-aminopyridine was neutralized by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca²⁺ channel blockers, ω-conotoxin GVIA, ω-conotoxin MVIIC or ω-conotoxin GVIA plus ω-conotoxin MVIIC, were greater than those caused by the Ca²⁺ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and ω-conotoxin GVIA or CPA and ω-conotoxin MVIIC were present was also greater than the inhibition by the Ca²⁺ channel blockers alone. The presence of both ω-conotoxin GVIA and ω-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [

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]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A₁ receptor activation partly inhibits P/Q- and other non-identified types of Ca²⁺ channels.     

Differential effects of chronic imipramine and fluoxetine on basal and amphetamine-induced extracellular dopamine levels in rat nucleus accumbens

The effect of chronic treatment with the tricyclic antidepressant drug, imipramine (10 mg/kg per day), the selective serotonin (5-HT) reuptake inhibitor, fluoxetine hydrochloride (10 mg/kg per day), and vehicle, in drinking water for 24–28 days followed by 3–5 days withdrawal, on extracellular dopamine levels was studied in rat nucleus accumbens by in vivo microdialysis. Basal extracellular dopamine levels in the nucleus accumbens were increased after chronic imipramine (12.7±1.5 fmol/20 μl per 30 min, P=0.019), and moderately decreased after chronic fluoxetine (6.5±0.6, P=0.047), as compared to the vehicle controls (9.1±0.7), determined by one-way analysis of variance (ANOVA). Repeated measure ANOVA indicated that the

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-amphetamine sulfate (0.5 mg/kg, s.c.)-induced increase in extracellular dopamine levels in the nucleus accumbens was potentiated after chronic imipramine (P=0.002), but unchanged after chronic fluoxetine (P=0.83). The difference in the effect of amphetamine could be influenced by the significant differences in basal levels. However, these results were also confirmed by analysis of the net area under the curve (net-AUC) for a 180-min period (six samples): for chronic imipramine (337±45 fmol/180 min, P=0.005) and chronic fluoxetine (249±38, P=0.57), as compared to the vehicle controls (178±29), determined by one-way ANOVA. We suggest that the effect of treatment with these agents on mesolimbic dopamine is unlikely to be involved in their shared antidepressant action, but may be relevant to other aspects of the therapeutic profile of these two drugs, e.g. the switch into mania which is more common after treatment with imipramine than fluoxetine and exacerbation of positive symptoms in patients with schizophrenia or schizoaffective disorder.     

Characteristics of the NMDA receptor modulating hypoxia/hypoglycaemia-induced rat striatal dopamine release in vitro

We investigated the functional characteristics of the NMDA receptor that modulates hypoxia/hypoglycaemia-induced striatal dopamine release. Dopamine release was detected by fast cyclic voltammetry in rat neostriatal slices. Four variables were measured: T_on — time from initiation of hypoxia/hypoglycaemia to the onset of dopamine release, T_pk — time from onset to maximum, δDA/δt — rate of dopamine release and DA_max — maximum extracellular dopamine concentration. In controls, T_on=164.9±1.7 s, T_pk=20.9±0.9 s, δDA/δt=5.31±0.44 μM/s and DA_max=79.1±2.5 μM (means±S.E.M., n=203). Cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755, 20 μM) lengthened, while N-methyl-

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-aspartate (NMDA) (100 μM) shortened T_on. (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK 801, 1 and 10 μM) and dextromethorphan (10 and 100 μM) increased T_pk and decreased DA_max. Neither glycine (100 μM), 7-chlorokynurenic acid (50 μM) nor 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione (ACEA 1021, 100 μM) had any effect although 7-chlorokynurenic acid blocked the effect of NMDA. Increasing [Mg²⁺] from 1.3 to 3.7 mM, increased T_pk and decreased δDA/δt. Dithiothreitol (1 mM) accelerated T_on while 5,5-dithio-bis-(2-nitrobenzoic acid) (1 mM) delayed T_on. Neither drug affected T_pk, DA_max or δDA/δt. Neither spermidine (100 μM) nor arcaine (100 μM) affected T_on, T_pk or δDA/δt although arcaine decreased DA_max. In conclusion, hypoxia/hypoglycaemia-induced dopamine release was influenced by an NMDA receptor although modulation of the glycine recognition site of the receptor was ineffective, as were agents acting at polyamine modulatory zones. These findings highlight differences between recombinant and native NMDA receptors and suggest caution in extrapolating molecular biology to functional studies.     

Evaluation of organic nephrotoxins in rabbit renal cortical slices

An in vitro cortical-slice system was used to assess the toxicity of several organic nephrotoxins that require transport and/or bioactivation in order to induce toxicity. The toxins cephaloridine, hexachlorobutadiene,

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and gentamicin all produce site-specific proximal tubular injury when administered in vivo. Damage was assessed in vitro by observing alterations in intracellular potassium, intracellular lactate dehydrogenase, and organic anion and cation accumulation. Histopathology was studied to assess the localization of injury. All four compounds produced dose- and time-dependent decreases in the biochemical parameters as well as site-specific lesions in the S₃ region. The results illustrate the usefulness of renal cortical slices in acute studies of organic nephrotoxins.     

Synthesis and antiviral properties of carbocyclic 3′-oxa-2′,3′-dideoxyguanosine and its 7-deazaguanosine analogue

To evaluate analogues of the antiviral agent (R)-9-(3,4-dihydroxybutyl)guanine in which the side-chain C-3 hydroxyl oxygen is part of a five-membered ring, carbocyclic 3′-oxa-2′,3′-dideoxyguanosine (4) and carbocyclic 3′-oxa-2′,3′-dideoxy-7-deazaguanosine (5) have been synthesized in 17 and 14 steps, respectively, from

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. Compounds 4 and 5 and their 6-chloro precursors were evaluated against a wide variety of DNA and RNA viruses. Only 4 showed any marginal activity and this was limited to HSV-1 and HSV-2. Even though 4 was less potent towards these latter two viruses than acyclovir, its mechanism and target of action is proposed to resemble that of acyclovir. The only toxicity observed for these compounds was observed in the cell growth assay with human embryonic lung cells.     

Thermospray liquid chromatography/mass spectrometry study of diastereomeric isoindole derivatives of amino acids and amino acid amides

A thermospray liquid chromatography/mass spectrometry (TSP-LC/MS) method is described for determination of the enantiomeric excess of -amino acids and -amino acid amides as their

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(OPA/NAC) derivatives.

The source temperature is an important factor in optimizing the sensitivity of the TSP-LC/MS analysis, whereas the repeller voltage is of minor importance. On-column mass spectra were acquired for the OPA/NAC derivatives of several -amino acids and -amino acid amides. For the main fragment ions, mass spectra fragmentation pathways are proposed. The applicability of the method is demonstrated by means of the enantiomeric excess determination of valine in a sample from an enzymatic hydrolysis experiment.

Using single ion monitoring, the detection limit of -valine in the presence of excess -valine is 10 pmol. The present TSP-LC/MS method is useful for validating the results obtained from LC/UV or LC/fluorescence methods for the enantiomeric excess determination of -amino acids and -amino acid amides.     

The lethal principle of Poa huecu (coiron blanco): a plant indigenous to Argentina

, , , and

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. The lethal principle of Poa huecu (Coiron Blanco): a plant indigenous to Argentina. Toxicon 27, 1251–1262, 1989.—The lethality of Poa huecu, a plant toxic to cattle and sheep, was followed by injection of chromatographic fractions in mice. The lethal aqueous extract was administered i.p. to Rockland mice of either sex and produced motor incoordination, transient ataxia, rough hair coat, tremors and muscle contractions and, occasionally, blindness. Doses greater than 1.5 g/kg mouse were always lethal. Fractionation of this lethal extract included dialysis, column chromatography on Sephadex G-25 and fractional precipitation with ethanol. Precipitates obtained with 70% and 85% ethanol were further purified on a DEAE-cellulose column. Eight fractions were obtained, each was injected into mice. Only fractions 3–6 were toxic. Fraction 3 produced slight hepatosis and hyperemia in the liver and gliosis in the brain. None of the other tissues exhibited histological lesions. Fractions 4 and 5 caused death of all animals within 30 min to 4 hr after injection. Polyacrylamide gel electrophoresis and acid hydrolysis showed that fractions 4 and 5 contained a glycoprotein of nearly the same mol. wt (67,000–94,000). Microscopic pathology in the mice treated with the lethal glycoprotein of fraction 4 included hyperemia in the kidneys, megakaryocytes in the spleen, slight hepatosis and focal coagulative necrosis with nuclear pyknosis and karyonexis in the liver, gliosis, intracellular brain edema with axon degeneration and swollen astrocytes in the brain. These brain injuries may relate to the motor incoordination of cattle that causes a delayed righting reflex. The major monosaccharides of the lethal glycoprotein are glucose and mannose, while rhamnose, arabinose, xylose and galactose are present in low percentages. Proline and the acidic amino acids (glutamic and aspartic acids) are the most abundant in the peptidic residue.     

Effects of natriuretic peptides on ventricular myocyte contraction and role of cyclic GMP signaling

Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) act through different receptors and at different potencies to affect cardiac myocyte function. We tested the hypothesis that these three peptides would differentially reduce cardiomyocyte function through their effects on the cyclic GMP signaling pathway. Rabbit ventricular myocytes were isolated and stimulated by electrical field stimulation. Cell function was measured using a video edge detector. ANP BNP or CNP at 10⁻⁹, 10⁻⁸, 10⁻⁷ M were added to the myocytes. Intracellular cyclic GMP was determined using a radioimmunoassay in the absence or presence of ANP, BNP or CNP. All natriuretic peptides decreased myocyte contractility in a similar concentration dependent manner. Myocyte percentage shortening was significantly decreased with all peptides at 10⁻⁷ M compared with baseline (ANP from 5.4±0.4 to 3.9±0.2%; BNP from 5.0±0.2 to 3.5±0.1%; CNP from 5.6±0.3 to 4.0±0.3%). Maximum rate of shortening and relaxation were also decreased similarly and significantly. Intracellular cyclic GMP was significantly increased in myocytes treated with ANP, BNP or CNP (Baseline 1.0±0.2, ANP 2.1±0.2, BNP 2.3±0.3, CNP 2.0±0.2 pmol/10⁵ myocytes). Furthermore, inhibition of the cyclic GMP protein kinase with KT5823 caused a reversal in the functional effects of CNP. We concluded that all natriuretic peptides had similar negative effects on ventricular myocyte function and their effects were accompanied by increased cyclic GMP. Blockade the effect of CNP by a cyclic GMP protein kinase inhibitor demonstrated that effects were mediated through the cyclic GMP signaling pathway.     

C-type natriuretic peptide inhibits rat mesangial cell proliferation by a phosphorylation-dependent mechanism

We studied the effects of C-type natriuretic peptide (CNP) on rat cultured mesangial cell proliferation. (1) Exposure to CNP (10 nM–1 μM for 72 h) inhibited [³H]thymidine incorporation into mesangial cells in a concentration-dependent manner. Atrial natriuretic peptide (1 nM–1 μM), a peptide related to CNP, also decreased [³H]thymidine incorporation into these cells in a concentration-dependent manner. (2) Both CNP (10 nM-1 μM) and atrial natriuretic peptide (10 nM-1 μM) also decreased mesangial cell number. (3) The cyclic GMP analog, 8-bromo-cyclic GMP (100 μM and 1 mM), mimicked the inhibitory effects of CNP and atrial natriuretic peptide on [³H]thymidine incorporation into mesangial cells, whereas inhibitors of protein kinase C, protein kinase A, and protein kinase G reduced the effect of both natriuretic peptides. Moreover, the phoshpatase inhibitor, calyculin A, increased [³H]thymidine incorporation into mesangial cells. (4) CNP and atrial natriuretic peptide decreased interleukin-1-, interleukin-6-, platelet derived growth factor-, angiotensin II-induced [³H]thymidine incorporation into mesangial cells. These results suggest that CNP exerts inhibitory effects on mesangial cell proliferation and that this effects depend on protein phosphorylation pathways. Received: 17 March 1997 / Accepted: 29 August 1997     

Type-C natriuretic peptide prevents development of experimental atherosclerosis in rabbits

1. We investigated the effect of local administration of type-C natriuretic peptide (CNP) on the endothelial dysfunction and development of an atheroma-like neointima induced by a peri-arterial collar in rabbits. 2. Peri-arterial collars were placed on both common carotid arteries allowing local treatment of the collared region with either CNP (10 micromol/L) or saline. After 7 days, uncollared (control) and collared sections were taken from both arteries for pharmacological and morphological analysis. 3. Application of the collar markedly attenuated (P < 0.05) endothelium-dependent vasorelaxation induced by acetylcholine (ACh); inhibition of 5-hydroxytryptamine contraction was 80+/-5% in control sections compared with 44+/-4% in collared sections from the same arteries. Local infusion of CNP (10 micromol/L) into the collar restored ACh-induced vasorelaxation (74+/-3% from collared arteries + CNP vs 77+/-2% from control sections from the same arteries). 4. Type-C natriuretic peptide treatment also reduced (P < 0.05) intimal thickening compared with contralateral collared arteries (intima/media ratio 0.06+/-0.01 vs 0.16+/-0.01). 5. These results provide evidence that locally administered CNP is effective in preventing the endothelial dysfunction and development of a neointima in this model.     

脂质体携载C型利钠利尿肽对血管效应的影响

目的　观察脂质体携载Ｃ 型利钠利尿肽（ＣＮＰ） 对大鼠主动脉血管舒张、平均动脉血压和内皮素（ＥＴ） 刺激主动脉平滑肌细胞增殖的影响。方法　主动脉环灌流测定血管张力;平滑肌细胞培养;［３Ｈ］ ＴｄＲ参入测定ＤＮＡ合成;反相蒸发法制备脂质体。结果　脂质体携载ＣＮＰ的舒张主动脉和降低平均动脉压作用显著强于单用ＣＮＰ。ＥＴ刺激平滑肌细胞增殖,使ＶＳＭＣ［３Ｈ］ ＴｄＲ 参入量增加１－８ 倍。１０ －８ 、１０－ ７ 、１０－ ６ ｍｏｌ·Ｌ－１ ＣＮＰ可使ＥＴ 刺激［３Ｈ］ ＴｄＲ参入量分别下降２％ （ Ｐ＞ ０－０５）, ２１ ％ （ Ｐ＜ ０－０５） 和５５％ （ Ｐ＜０－０１） ,而同样浓度的脂质体携载ＣＮＰ 使ＥＴ 刺激ＶＳＭＣ［３Ｈ］ ＴｄＲ参入量分别下降５６％ （ Ｐ＜ ０－０１）,５９％ （ Ｐ＜０－０１） 和６６ ％ （ Ｐ＜ ０－０１）, 其作用强于ＣＮＰ。脂质体和ＣＮＰ对［３Ｈ］ ＴｄＲ 参入量与对照组相比差异无显著性。结论　脂质体携载ＣＮＰ的舒张血管、降低平均动脉压和抑制ＥＴ 刺激平滑肌细胞增殖作用强于ＣＮＰ     

Effects of adrenomedullin on rat cerebral arterioles

The effects of adrenomedullin on isolated rat intracerebral arterioles were investigated and compared with those of calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin produced dose-dependent vasodilation (maximum dilation 27.1±2.1% at 3×10⁻⁷ M, median effective dose (EC₅₀) 1.6×10⁻⁹ M). CGRP produced similar vasodilation (19.8±4.1%) at 10⁻⁷ M with a lower EC₅₀ of 2.8×10⁻¹¹ M. Amylin did not cause vasodilation at concentrations up to 10⁻⁶ M. Adrenomedullin-induced vasodilation was significantly suppressed by CGRP-(8–37). These data suggest that adrenomedullin is a potent vasodilator for arterioles in the cerebral microcirculation that acts through CGRP receptors.     

MODULATORY EFFECTS OF TYPE-C NATRIURETIC PEPTIDE ON SYMPATHETIC COTRANSMISSION IN THE RAT ISOLATED TAIL ARTERY

1. Rat type-C natriuretic peptide (CNP) has been studied for its effects on the neurogenically induced overflow of adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), adenosine 5-monophosphate (AMP), adenosine (ADO) and noradrenaline (NA) in endothelium-free segments of rat isolated tail artery. The overflow of each was evoked by electrical field stimulation (EFS) of 0.5 ms pulses at 8 Hz for 3 min and the amount of ATP, ADP, AMP and ADO was quantified by high-performance liquid chromatography (HPLC)-fluorescent detection, while the amount of NA was quantified by HPLC-electrochemical detection. 2. Type-C natriuretic peptide (100 nmol/L) was found to cause a significant reduction of the overflow of all adenine purines and NA. However, at lower concentrations (1 and 10 nmol/L), CNP caused a significant reduction of the overflow of NA but did not change ATP overflow. 3. The overflow of ADP, AMP and ADO was significantly reduced by either concentration of CNP, so that the ratio ATP:ADP was diminished from 1:2 in controls to 1:1 after 1 nmol/L CNP and to 1:1.2 after 10 nmol/L CNP. 4. The production of inorganic phosphate (P_i) in response to the exogenous application of ATP was significantly reduced by 1,10 or 100 nmol/L CNP. 5. Type-C natriuretic peptide exerts neuromodulatory effects on the neurogenically induced release of the cotransmitters ATP and NA in rat tail artery, consisting of an inhibition of the release of both ATP and NA. This effect is accompanied by inhibition of the breakdown of ATP by ecto-ATPases. Either effect results in apparent CNP-induced differential modulation of the overflow of the cotransmitters ATP and NA.     

NEUTRAL ENDOPEPTIDASE 3.4.24.11 INHIBITION POTENTIATES THE INHIBITORY EFFECTS OF TYPE-C NATRIURETIC PEPTIDE ON LEUKOTRIENE D4-INDUCED AIRWAY CHANGES

1. Microvascular leakage, a primary feature of inflammation, is well known for worsening the asthmatic condition. Gene expression of and a specific receptor for type-C natriuretic peptide (CNP), initially considered a neuropeptide, have been detected in the human vascular wall and secretion of CNP from vascular endothelial cells has recently been demonstrated. These facts suggest the presence of a vascular natriuretic peptide system and led us to expect that CNP may act beneficially on airway microvascular leakage in asthma. In the present study, we investigated the effects of CNP against leukotriene (LT) D4 -induced airway microvascular leakage and bronchocon-striction and how these effects were potentiated by thiorphan, a potent neutral endopeptidase 3.4.24.11 (NEP) inhibitor. 2. Anaesthetized male guinea-pigs, ventilated via a tracheal cannula, were placed into a plethysmograph for 10 min, in order to measure pulmonary mechanics and mean blood pressure, after challenge with 2 μg/kg LTD₄ and then the extravasation of 20 mg/kg Evans blue dye into airway tissue was investigated to indicate and evaluate microvascular leakage. 3. Intravenous administration of CNP (100, 300 and 1000 μg/kg) significantly inhibited the LTD₄-induced microvascular leakage and bronchoconstriction in a dose-dependent manner. These inhibitory effects were enhanced by pretreatment with 20 mg/kg thiorphan, suggesting the important role of NEP in the pulmonary metabolism of CNP. 4. We believe that these results are encouraging for the further investigation of the therapeutic applications of exogenous CNP in asthma.     

C‐type natriuretic peptide regulation of guanosine‐3′,5′‐cyclic monophosphate production in human endothelial cells

1 In vascular smooth muscle cells, relaxant actions of guanosine‐‐3′,5′‐cyclic monophosphate (cGMP) are well recognized, but there is increasing evidence that cGMP also plays regulatory roles in vascular endothelium. However, the autacoid and endocrine mechanisms controlling cGMP production in endothelium are not well understood. The objective of these studies was to examine the mechanisms of cGMP accumulation in human umbilical vein endothelial cells (HUVEC) in response to natriuretic peptides. 2 Expression in HUVEC of natriuretic peptide receptors, particulate guanylyl cyclases (GC)‐A and GC‐B, was confirmed by RT‐PCR and Western blot analysis. 3 In the presence of the phosphodiesterase inhibitor IBMX 500 μm , 3 h incubation of HUVEC with B‐type natriuretic peptide (BNP) (preferential GC‐A agonist) or C‐type natriuretic peptide (CNP) (preferential GC‐B agonist) stimulated concentration‐dependent increases in cGMP production. At 10 and 100 nm , we observed two to three‐fold greater potency of CNP compared to BNP. 4 In the absence of IBMX, CNP‐stimulated cGMP accumulation was significantly less than cGMP accumulation in response to sodium nitroprusside 1 mm . This greater sensitivity of GC‐B‐derived cGMP to phosphodiesterases suggests compartmentalization of two pools of cGMP from particulate and soluble guanylyl cyclases. 5 Although CNP 100 nm and 1 μm was observed to increase nitrite + nitrate (stable metabolites of NO) production in HUVEC two‐fold above basal level, the soluble guanylyl cyclase inhibitor ODQ 10 μm did not significantly modify CNP‐stimulated cGMP accumulation suggesting that endothelial actions of CNP may be NO‐independent. 6 In conclusion, these studies indicate functional signaling by natriuretic peptides in endothelial cells, supporting possible roles of these mediators in regulating endothelial cell function.