Pilar D'Ocon

Discrepancies Between Nitroglycerin and NO-Releasing Drugs on Mitochondrial Oxygen Consumption, Vasoactivity, and the Release of NO

It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.

Selective inhibition of calcium entry induced by benzylisoquinolines in rat smooth muscle

Abstract— The mechanism of relaxant activity of six benzylisoquinolines was examined in order to determine the minimal structural requirements that enable these compounds to have either a non‐specific action like papaverine or an inhibitory activity on calcium entry via potential‐operated channels. All the alkaloids tested totally or partially relaxed KCl‐depolarized rat uterus and inhibited oxytocin‐induced rhythmic contractions. Only glaucine and laudanosine inhibited K+‐induced uterine contractions more than oxytocin‐induced uterine contractions. In Ca+‐free medium, sustained contractions induced by oxytocin or vanadate were relaxed by the alkaloids tested except for glaucine and laudanosine indicating no inhibitory effect on intracellular calcium release. Those alkaloids containing an unsaturated heterocyclic ring (papaverine, papaverinol, papaveraldine, N‐methylpapaverine and dehydropapaverine) exhibited a more specific activity than those with a tetrahydroisoquinoline ring.

Regulation of Oxygen Distribution in Tissues by Endothelial Nitric Oxide

Nitric oxide (NO) decreases cellular oxygen (O2) consumption by competitively inhibiting cytochrome c oxidase. Here, we show that endogenously released endothelial NO, either basal or stimulated, can modulate O2 consumption both throughout the thickness of conductance vessels and in the microcirculation. Furthermore, we have shown that such modulation regulates O2 distribution to the surrounding tissues. We have demonstrated these effects by measuring O2 consumption in blood vessels in a hypoxic chamber and O2 distribution in the microcirculation using the fluorescent oxygen-probe Ru(phen)32+. Removal of NO by physical or pharmacological means, or in eNOS−/− mice, abolishes this regulatory mechanism. Our results indicate that, in addition to its well-known effect on the regulation of vascular tone, endothelial NO plays a major role in facilitating the distribution of O2, an action which is crucial for the adaptation of tissues, including the vessel wall itself, to hypoxia. It is possible that changes in the distribution of O2 throughout the vessel wall may be implicated in the origin of vascular pathologies such as atherosclerosis.

Pathological role of a constitutively active population of α1D-adrenoceptors in arteries of spontaneously hypertensive rats

The role of a constitutively active population of α1D‐adrenoceptors was analysed in arteries obtained from spontaneously hypertensive rats (SHR) and controls (WKY) divided into three groups: young prehypertensive, adult hypertensive, and adult animals chronically treated with captopril (50 mg kg−1 per day orally) in order to prevent the hypertensive state. In adult SHR, a significant increase in BMY 7378 potency (not in prazosin potency) was observed in aorta, mesenteric artery, and the first and second branches of the small mesenteric arteries with respect to WKY rats. This difference was not observed in iliac and tail arteries, which suggests an increased functional role of α1D‐adrenoceptors only in some vessels of SHR. The increase in the resting tone (IRT) observed in absence of agonist, inhibited by BMY 7378, that represents the constitutively active population of α1D‐adrenoceptors, was also significantly greater in aorta and mesenteric artery from adult SHR. In young and captopril treated adult animals, no differences between strains with respect to BMY 7378 potency, or IRT were observed. The increase in the functional role of α1D‐adrenoceptors and their constitutive activity observed in hypertension is prevented by captopril treatment. The pathological consequence of this change is the slower rate of recovery of the basal tone after removal of an adrenergic stimulus, observed in vessels from hypertensive animals that had shown an increase in the functionality of constitutively active α1D‐adrenoceptors. This change was not observed in prehypertensive or captopril treated animals.

Functional evidence of inverse agonism in vascular smooth muscle.

1 In the present study, depletion of internal Ca2+ stores sensitive to noradrenaline (1 μm) in rat aorta, is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, in our experimental conditions, to activate the contractile proteins. This induces an increase in the resting tone that constitutes, the first functional evidence of this Ca2+ entry. 2 The fact that methoxamine (100 μm) reproduces the same processes as noradrenaline but clonidine (1 μm) does not, indicates that α1‐adrenoceptor activation is related to the increase in the resting tone observed after depletion of adrenoceptor‐sensitive internal Ca2+‐stores. 3 Benoxathian and WB 4101 (α1A‐ and α1D‐adrenoceptor antagonists) selectively inhibit, in a concentration‐dependent manner, this mechanical response observed in absence of the agonist, which suggests that these agents can act as inverse agonists and provide a functional model for studying this phenomenon. Since chloroethylclonidine (100 μm) has no effect on this response, the participation of α1B‐adrenoceptors can be ruled out. 4 Contractile responses to noradrenaline (1 μm) in Ca2+‐free medium were selectively blocked by chloroethylclonidine. This suggests that the response to noradrenaline in Ca2+‐free medium mainly depends on the activation of the α1B‐adrenoceptor subtype.

Multiple actions of glaucine on cyclic nucleotide phosphodiesterases, α1‐adrenoceptor and benzothiazepine binding site at the calcium channel

1 In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of α‐adrenoceptors, by use of [3H]‐(+)‐cis‐diltiazem, [3H]‐nitrendipine and [3H]‐prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2 Contraction evoked by noradrenaline (1 μm) or depolarizing solution (60 mm KCl) were inhibited in a concentration‐dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA‐induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl‐induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3 In Ca2+‐free solution, prazosin (0.1 μm) and glaucine (0.1 mm) inhibited the contraction evoked by NA; diltiazem (0.1 mm) diminished this contraction whereas nifedipine (1 μm) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mm) significantly inhibited the contractions evoked by NA or caffeine in Ca2+‐free medium. 4 Glaucine and papaverine show affinity at the [3H]‐prazosin binding site and at the benzothiazepine binding site of the Ca2+‐channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex. Glaucine exerts some selectivity as an inhibitor of [3H]‐prazosin binding as opposed to [3H]‐(+)‐cis‐diltiazem binding while papaverine appears to have approximately equal affinity in this respect. 5 This study confirms the presence of four phosphodiesterase (PDE) activities in bovine aorta: a calmodulin‐activated PDE (CaM‐PDE type I) which hydrolyzed preferentially guanosine 3′:5′‐cyclic monophosphate (cyclic GMP); a cyclic GMP selective form (cGMP‐PDE type V); and two low Km adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) PDEs that are insensitive to the stimulatory effect of CaM, one of which was inhibited by cyclic GMP (CGI‐PDE, type III) and the other by rolipram (cAMP‐PDE, type IV). Glaucine selectively inhibits one of the two forms of Ca2+‐independent low Km cAMP‐PDE, the type IV. In contrast, papaverine exerts a non‐selective inhibitory effect upon all PDE forms. 6 The present work provides evidence that glaucine, a benzyltetrahydroisoquinoline alkaloid, has interesting properties as an α1‐adrenoceptor antagonist, calcium entry blocker (through the benzothiazepine recognition site in the calcium channel) and as a selective inhibitor of the rolipram‐sensitive cAMP‐PDE, type IV PDE.

Different β-adrenoceptor subtypes coupling to cAMP or NO/cGMP pathways: implications in the relaxant response of rat conductance and resistance vessels

To analyse the relative contribution of β1‐, β2‐ and β3‐adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways.

Mechanism of the cardiovascular activity of laudanosine: comparison with papaverine and other benzylisoquinolines.

1 The activity of (±)‐laudanosine, a benzyltetrahydroisoquinoline alkaloid, was investigated in pithed rats and rat isolated aorta. Its effects on [3H]‐prazosin, [3H]‐(+)‐cis‐diltiazem and [3H]‐nitrendipine binding to rat cerebral cortical membranes, and on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were investigated. 2 The dose‐response curve to methoxamine (3–300 μg kg−1, i.v.) in normotensive pithed rats was shifted to the right by (±)‐laudanosine, 3 and 6 mg kg−1. 3 (±)‐Laudanosine inhibited in a concentration‐dependent manner the contractile responses evoked by noradrenaline (NA 1 μm), depolarizing solution (KC1 80 μm) or depolarizing solution plus phentolamine (10 μm) in rat isolated aorta. The alkaloid appeared to be more potent against NA‐induced contractions. 4 In Ca2+‐free solution, (±)‐laudanosine (100 μm) inhibited the contraction evoked by NA and did not modify the phasic contractile response evoked by caffeine. The alkaloid did not modify the refilling of the intracellular Ca2+‐stores sensitive to NA or caffeine. 5 (±)‐Laudanosine inhibited [3H]‐prazosin binding to cortical membranes and also inhibited [3H]‐(+)‐cis‐diltiazem but with a lower potency. [3H]‐nitrendipine binding was not affected by laudanosine. 6 (±)‐Laudanosine does not have a significant effect on the different forms of PDEs isolated from bovine aorta. In contrast, compounds structurally related to this alkaloid such as papaverine and its derivatives, had a non‐selective or more specific inhibitory effect on these PDE forms. These differences can be explained on the basis of their structural features: the planarity of the isoquinoline ring (papaverine) facilitates the interaction with receptor sites, and the different position of the benzyl group does not modify the activity unless this position leads to the presence of a chiral centre (laudanosine). 7 These results suggest that (±)‐laudanosine has a selective activity as an α1‐adrenoceptor blocker. Its lack of action on different PDE forms provides us with information about a group of benzylisoquinolines that with small structural changes show a different effect on PDE‐forms isolated from vascular smooth muscle.

Inhibition of calcium entry induced by cularines and isocrasifoline in uterine smooth muscle

The effects of nifedipine, papaverine and four benzylisoquinoline alkaloids (cularine, cularidine, celtisine and isocrasifoline) were studied in isolated rat uterus in order to clarify the mechanism of their relaxant action. All the compounds tested completely relaxed KCl-induced contractions and totally or partially inhibited oxytocin-induced rhythmic contractions. Only papaverine acted intracellularly, promoting relaxation of contractile responses induced by oxytocin or vanadate in a Ca(2+)-free medium. In spite of the structural relationship between papaverine and the other alkaloids, the mechanism of their relaxant action is not the same. The activities of cularine derivatives and of isocrasifoline were similar to that of nifedipine.

The impact of alpha1-adrenoceptors up-regulation accompanied by the impairment of beta-adrenergic vasodilatation in hypertension.

In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of β-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of α1A, α1B, α1D, β1, β2, and β3-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of β1-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. α1D- and β3-Adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (α1D-ARs are the most sensitive to agonists, and β3-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the α1D subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.