Indomethacin promotes nitric oxide function in the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2) in concert with nitric oxide (NO) and carbon monoxide (CO). Accordingly, we have previously found that NO activity increases upon deletion of either COX. Here, we have examined whether COX inhibition by indomethacin mimics COX deletion in promoting NO. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type (WT) and eNOS-/-, near-term mouse foetuses. Indomethacin was given p.o. to the mother as single (acute treatment) or repeated (daily for 3 days; chronic treatment) doses within a therapeutic range (2 mg kg(-1)). KEY RESULTS: Indomethacin promoted eNOS mRNA expression in the WT ductus. Coincidentally, the drug enhanced the contraction of the isolated ductus to the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, and its effect augmented with the length of treatment. No such enhancement was seen with the eNOS-/- ductus. Chronic indomethacin also increased, albeit marginally, the contraction of the WT ductus to the CO synthesis inhibitor, zinc protoporphyrin. Whether given acutely or chronically, indomethacin induced a little narrowing of the ductus antenatally and had no effect on postnatal closure of the vessel. CONCLUSIONS AND IMPLICATIONS: We conclude that activation of NO and, to a much lesser degree, CO mechanisms is an integral part of the indomethacin effect on the ductus. This relaxing influence may oppose the contraction from PGE(2) suppression and could explain the failures of indomethacin therapy in premature infants with persistent duct.

Interactions between NO, CO and an endothelium-derived hyperpolarizing factor (EDHF) in maintaining patency of the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2), possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO-2) and COX1 or COX2. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type and gene-deleted, near-term mouse fetuses. KEY RESULTS: N(G)-nitro-L-arginine methyl ester (L-NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L-NAME contraction was not modified by HO-2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin-contracted ductus similarly in wild-type and eNOS-/- or iNOS-/-. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K(+) increase or K(+) channel inhibition. In vivo, the ductus was patent in wild-type and NOS-deleted fetuses. Likewise, no genotype-related difference was noted in postnatal closure. CONCLUSIONS AND IMPLICATIONS: NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BK-induced relaxation in the absence of PGE(2). However, in the absence of PGE(2), NO and CO, BK induces a relaxant substance behaving as an endothelium-derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE(2) and NO being preferentially coupled for reciprocal compensation.

Role of cyclooxygenases 1 and 2 in the modulation of neuromuscular functions in the distal colon of humans and mice.

BACKGROUND: Cyclooxygenase isoforms (COX-1, COX-2) may exert differential regulatory actions on enteric motor functions under normal or pathological conditions. AIMS: To examine the occurrence and functions of COX-1 and COX-2 in the neuromuscular compartment of normal distal colon using human and murine tissue. METHODS: Gene expression (human, mouse), protein expression (human), gene deletion (mouse), and the effects of dual and isoform specific COX inhibitors on in vitro motility (human, mouse) were investigated. RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) showed mRNA expression of COX-1 and COX-2 in human and wild-type mouse colonic muscle whereas only COX-2 or COX-1 was detected in COX-1 or COX-2 knockout animals. Immunohistochemistry localised both isoforms in neurones of myenteric ganglia, COX-1 in circular layer myocytes, and COX-2 in longitudinal muscle. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor), or DFU (COX-2 inhibitor) enhanced atropine sensitive electrically induced contractions of human longitudinal muscle. The most prominent actions were recorded with indomethacin or SC-560 plus DFU. These results were confirmed under pharmacological blockade of non-cholinergic nerves. Atropine sensitive contractions evoked by carbachol in the presence of tetrodotoxin were enhanced by indomethacin or DFU but not by SC-560. In wild-type mice, contractile responses to electrical stimulation were enhanced by indomethacin, SC-560, or DFU. SC-560 potentiated electrically induced contractions in COX-2, but not COX-1, knockout mice. In contrast, DFU enhanced the contractions elicited by electrical stimuli in COX-1, but not in COX-2, knockout mice. CONCLUSIONS: These results indicate that COX-1 and COX-2 are expressed in the neuromuscular compartment of normal human colon where they modulate cholinergic excitatory control of colonic motility at prejunctional and postjunctional sites, respectively.

Cyclooxygenase-1 and cyclooxygenase-2 in the mouse ductus arteriosus: individual activity and functional coupling with nitric oxide synthase.

1. Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E(2), conceivably in concert with nitric oxide (NO). Local PGE(2) formation is sustained by cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near-term mouse ductus and the possibility of COX deletion causing NO upregulation. 2. COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES. 3. The isolated ductus from wild-type and COX1-/- mice contracted promptly to indomethacin (2.8 micro M). Conversely, the contraction of COX2-/- ductus to the same inhibitor started only after a delay and was slower. 4. N(G)-nitro-L-arginine methyl ester (L-NAME, 100 micro M) weakly contracted the isolated wild-type ductus. Its effect, however, increased three- to four-fold after deleting either COX, hence equalling that of indomethacin. 5. In vivo, the ductus was patent in all mice foetuses, whether wild-type or COX-deleted. Likewise, no genotype-related difference was noted in its postnatal closure. 6. We conclude that the mouse ductus has a complete system for PGE(2) synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE(2) and NO can function synergistically in keeping the ductus patent.

Some structural changes on triazolyl-benzotriazoles and triazolyl-benzimidazolones as potential potassium channel activators. III.

This paper reports the synthesis and pharmacological evaluation of some compounds, obtained by structural modifications of 1,2,3-triazolyl-benzotriazoles and 1,2,3-triazolyl-benzimidazolones, which had shown activity as potential activators of the big-conductance calcium-activated potassium channels (BK(Ca)). Changes have concerned the introduction of a hinderer substituent in the 5-position of the benzimidazolone (4a, b) and benzotriazole (5a, b) rings, opening of the benzimidazolone ring (7) and substitution of the 1,2,3-triazole ring with a 2-hydroxyphenyl ring (10). Furthermore a series of 3-aryl-benzotriazin-4-one derivatives (13a-e) has been studied, which appears as a modification and/or combination of the benzimidazolone and benzotriazole rings. Only compound 10 shows interesting activity, while the other structural modifications either do not increase (compounds 4 and 5) or reduce (compounds 7 and 13) the pharmacological activity. However, these results provide useful information about structure-activity relationships.

Triazolyl-benzimidazolones and triazolyl-benzotriazoles: new potential potassium channel activators. II.

This paper reports the synthesis and pharmacological evaluation of a series of 5-substituted-triazolyl benzotriazoles (2a-f) and the corresponding series of 5-substituted-triazolyl-benzimidazolones (6a-f), as potential activators of the big-conductance calcium-activated potassium channels (BK(Ca)). The synthesis and structure demonstration of the stock compounds of the two series have been described in our previous works, as well as the common starting compounds 4-carboxamido-5-(4-substituted-2amino-anilino)-1,2,3-triazoles (1a-f). The triazolyl-benzotriazoles were obtained by diazotization, while the triazolyl-benzimidazolones were obtained by thermal intramolecular cyclization of ethoxycarbonylamino derivatives or directly with phosgene. Benzimidazolone compounds generally showed little effect whilst the compounds with a benzotriazole ring showed full efficacy, with vasorelaxing properties and potency parameters a little lower than that of the reference compound NS 1619. These effects were significantly reduced by an increased membrane depolarization. This depolarization-sensitive response is in agreement with the pharmacodynamic hypothesis of activation of potassium channels.

5-(4'-Substituted-2'-nitroanilino)-1,2,3-triazoles as new potential potassium channel activators. I.

By the hypothesised correlation with the large conductance Ca(++)-activated potassium channel (BK(Ca)) openers NS 004 and NS 1619, bearing a benzimidazolone ring, a series of new 5-(4'-substituted-2'-nitroanilino)-1,2,3-triazoles were synthesised and tested on in vitro isolated vascular preparation. The compounds were prepared starting from the appropriately substituted 2-nitro-phenylazides by 1,3-dipolar cycloaddition reaction to cyanoacetamide and following Dimroth isomerisation of the corresponding 1-arylsubstituted-5-amino-1,2,3-triazoles. The analogous 5-(4'-substituted-2'-amino-anilino)-1,2,3-triazoles were also prepared to assess the role of the nitro group in the pharmacophoric model. Almost all the nitro compounds showed a vasorelaxant activity on endothelium-denuded rat aortic rings with a potency comparable to that recorded for the reference compound NS 1619. Such a vasorelaxing activity was significantly reduced by the increase of the level of membrane depolarisation and by the potassium channel blocker 4-aminopyridine with a pharmacodynamic behaviour consistent with a potassium channel activation.

Ethnopharmacobotany in Tuscany: plants used as antihypertensives.

Among the more than 400 plants used in popular medicine in Tuscany, over 30 are used in the therapy of hypertension. For some of them their use is already known while for others there is no documentation. In this work we present the first results obtained from research carried out on antihypertensive plants belonging to Gentianaceae and in particular Gentiana kokiana.

Experimental and theoretical comparisons between the classical Schild analysis and a new alternative method to evaluate the pA2 of competitive antagonists.

The Schild analysis is undoubtedly the most frequently used powerful diagnostic tool to investigate the nature of an antagonist and, consequently, to evaluate its potency, often expressed as pA2. Nevertheless, different reasons often prevent the experimenter from applying this analysis, leading to use an inhibition curve for the antagonist and to evaluate its potency by means of several approaches, which are generally considered theoretically invalid. In a recent work, a new theoretical approach, mathematically analogous to the Schild one, has been shown. By means of a simplified experimental protocol based on an antagonist inhibition curve (following a control concentration-response curve for the agonist), this method allows a linear regression analysis, giving a slope value absolutely equivalent to the Schild slope and a reliable estimation of the pA2 of a competitive antagonist. In this paper, this new method has been compared with the Schild analysis, to determine the parameters of potency relative to well-known competitive antagonists, on different in vitro isolated preparations. In strips of guinea pig isolated gastric smooth muscle, pirenzepine antagonised the effects of bethanechol. In guinea pig isolated ileum, atropine blocked the contracturant effects of carbamylcholine, while in electrostimulated ileum segments, the inhibitory responses to alpha-methylnoradrenaline were reduced by idazoxan. Finally, in guinea pig isolated spontaneously beating atria, the negative inotropic effects of 5'-N-ethylcarboxamidoadenosine were antagonised by 8-cyclopentyl-1,3-dipropylxanthine. The parameters of potency, relative to all the above competitive antagonists and expressed as pA2, resulted almost equivalent, when calculated by the Schild analysis or by the alternative method. Furthermore, when tested also for the well-known irreversible alkylating agent dibenamine in rat aortic rings stimulated by noradrenaline, the alternative method furnished a profile of clear nonlinearity, unmasking the nature of the antagonism. Finally, the relationships between the results calculated by the alternative analysis or by the Schild analysis and different levels of computer-generated "random noise" (affecting the shape and the position of theoretical curves) were also evaluated, in order to know the robustness of the new method. The two methods proved reliable and almost equivalent in robustness, when applied with different levels of "random noise". These results confirm the Schild analysis as the most accurate tool to study antagonists, since this analysis can furnish the highest number of information and observations on the behaviour of an antagonist. Nevertheless, when limiting conditions prevent a classical Schild analysis and impose the use of an inhibition curve, the new method probably represents the most preferable experimental approach. Indeed, it allows to calculate the antagonist potency, after the evaluation of a slope parameter giving an important information about the possible nature of the antagonism.

Vascular effects of aqueous crude extracts of Artemisia verlotorum Lamotte (Compositae): in vivo and in vitro pharmacological studies in rats.

Artemisia verlotorum Lamotte (Compositae), growing in almost all the northern hemisphere, is used in folk medicine of some countries of Tuscany, Italy, as a remedy for hypertension. The pharmacological evaluation of the responses evoked by an aqueous dried extract of Artemisia verlotorum on the blood pressure of anaesthetized rats and on in vitro rat isolated aortae showed a marked, but transient, hypotensive activity. This effect was mediated by a strong vasodilator action, closely linked to the release of endothelial nitric oxide and to the nitric oxide-guanosine 3'-5'-cyclic monophosphate (cGMP) pathway, caused by a muscarinic receptor agonism.