M. Dolores Ivorra

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.

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.

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.

Selective action of two aporphines at α1-adrenoceptors and potential-operated Ca2+ channels

Abstract Contractions evoked by noradrenaline (1 μM) or a depolarizing solution of 60 mM KCI were concentration dependently depressed by the aporphine alkaloids (S)-boldine and (R)-apomorphine in rataorta. Both drugs had a greater inhibitory potency on the contraction elicited by noradrenaline. Dose-response curves for noradrenaline were shifted to the right in presence of (S)-boldine. (R)-Apomorphine acted by a complex mechanism at α 1 -adrenoceptors and its inhibitory effects was irreversible. The conformational features of these alkaloids may explain their different behaviour at α 1 -adrenoceptors. In Ca 2+ -free solution, the alkaloids inhibited the contraction evoked by noradrenaline but did not modify (apomorphine) or increase (boldine) the contractile response induced by caffeine. Both alkaloids interacted with [ 3 H]prazosin binding and with the benzothiazepine binding site of the Ca 2+ entry receptor complex but had no effect at the dihydropyridine binding site in the rat cerebral cortex. Both drugs showed some selectivity as inhibitors of [ 3 H]prazosin binding as opposed to [ 3 H]d-cis ditiazem binding. (R)-Apomorphine slightly inhibited one of the two forms of the Ca 2+ -independent, low K m cyclic AMP-phosphodiesterase (type IV), whereas it did not have a significant effect on the other phosphodiesterase forms. (S)-Boldine had negligible inhibitory effects on all phosphodiesterase forms. The present study provides evidence that (S)-boldine and (R)-apomorphine have interesting properties as Ca 2+ entry blockers (through the benzothiazepine receptor site in the Ca 2+ channel) and at α 1 -adrenoceptors.

CHARACTERIZATION OF TWO DIFFERENT CA2+ ENTRY PATHWAYS DEPENDENT ON DEPLETION OF INTERNAL CA2+ POOLS IN RAT AORTA

Abstract Ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM) produced a slow, sustained contractile response in rat aorta that only can be observed in Ca2+-containing solution. In Ca2+-free medium, no response to the drugs was obtained, which suggests that the contraction elicited in presence of Ca2+ is mainly due to the contribution of extracellular influx. This Ca2+ entry does not depend on the opening of dihydropyridine-dependent Ca2+-channels for nimodipine does not affect this.Noradrenaline (1 μM) induced a biphasic response in Ca2+-free medium that was mediated by two different Ca2+ compartments, one of which is common to caffeine (10 mM), and is also depleted by ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM). This compartment loses its Ca2+ content after long exposure (65 min) to Ca2+-free EDTA-containing solution and its refilling was also affected by the three agents tested. The other compartment depleted by noradrenaline, but not by caffeine, was also insensitive to ryanodine, thapsigargin and cyclopiazonic acid, and did not lose its Ca2+ after 65 min in Ca2+-free medium.Contractions induced by noradrenaline (1 μM) or caffeine (10 mM) in Ca2+-free medium were not affected by ryanodine, thapsigargin and cyclopiazonic acid when these agents were added 1 min before or during the response to each agonist. After depletion of internal Ca2+ stores sensitive to noradrenaline, an increase in the resting tone (IRT) of rat aorta was observed when Ca2+ was added again in absence of the agonist. This IRT was not affected by treatment with ryanodine, thapsigargin and cyclopiazonic acid, and represents a Ca2+ entry pathway dependent on the depletion of the noradrenaline-sensitive Ca2+ compartment. In conclusion, we can differentiate two Ca2+ entry pathways in rat aorta that depend on the previous depletion of two internal Ca2+ compartments: One corresponds to the classic capacitative Ca2+ entry model and is promoted by depletion of the internal pool sensitive to noradreanline, caffeine, ryanodine, thapsigargin and cyclopiazonic acid, the other is dependent only on depletion of an α1-adrenoceptor-sensitive Ca2+ pool.

Capacitative Ca2+ entry associated with α1-adrenoceptors in rat aorta

Abstract In rat aorta, depletion of internal Ca2+ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca2+ pools. This response cannot be repeated, suggesting a depletion of internal Ca2+ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca2+ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work.The fact that the addition of nimodipine (10–15–10–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca2+ influx through dihydropyridine-sensitive Ca2+ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10–10–10–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K+ channels to the mechanism of the IRT.The refilling of the two internal Ca2+ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca2+ compartment responsible for the tonic contraction induced by noradrenaline in Ca2+-free medium, whereas the refilling of the Ca2+ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca2+-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca2+ compartments sensitive to noradrenaline was inhibited.In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca2+-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca2+ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca2+ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca2+ channel. The Ca2+ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K2+ channels.

The effect of S‐(+)‐boldine on the α1‐adrenoceptor of the guinea‐pig aorta

1 The cardiovascular activity of S‐(+)‐boldine, an aporphine alkaloid structurally related to papaverine, was determined. The work includes functional studies on guinea‐pig isolated aorta contracted with noradrenaline, caffeine, KCl or Ca2+, and on guinea‐pig trachea contracted with acetylcholine or histamine. 2 S‐(+)‐boldine inhibited in a concentration‐dependent manner the contractile reponse evoked by noradrenaline (10 μm) in guinea‐pig aorta (IC50 = 1.4 ± 0.2 μm) while the KCl depolarizing solution (60 mM)‐ or the Ca2+ (1 mM)‐induced contractions were only partially affected by boldine up to 300 μm. In contrast, papaverine relaxed noradrenaline (NA), KCl or Ca2+ induced contractions showing similar IC50 values in all cases. S‐(+)‐boldine had a greater potency on the contraction elicited by NA whereas papaverine acted in a non‐selective manner. 3 S‐(+)‐boldine was found to be an α1‐adrenoceptor blocking agent in guinea‐pig aorta as revealed by its competitive antagonism of noradrenaline‐induced vasoconstriction (pA2 = 5.64 ± 0.08), and its potency was compared with that of prazosin (pA2 = 8.56 ± 0.24), a known potent α1‐adrenoceptor antagonist. In contrast, papaverine caused rightward shifts of the NA concentration‐response curves with depression of maximal response indicating that it acts as a non‐competitive antagonist. 4 Contraction of guinea‐pig aorta induced by caffeine (60 mM) in a Ca2+‐containing Krebs solution was not affected by a 60 min incubation period with different doses of S‐(+)‐boldine (1–300 μm). Papaverine inhibited partially this caffeine‐induced contraction at the maximal dose used (100 μm). 5 Inositol phosphates formation induced by noradrenaline (10 μm) in guinea‐pig thoracic aorta was inhibited by S‐(+)‐boldine (30 μm but not by papaverine (10 μm). 6 Contractions of guinea‐pig trachea caused by acetylcholine (100 μm) or histamine (10 μm) were not modified by S‐(+)‐boldine (0.1–100 μm). 7 These results provide evidence that S‐(+)‐boldine, an aporphine alkaloid, has interesting properties as an α1‐adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the α1‐adrenoceptor present in the guinea pig aorta.

Different Mechanism of Relaxation Induced by Aporphine Alkaloids in Rat Uterus

Abstract— We have examined the uterine relaxant action of three aporphine molecules (S‐glaucine, S‐boldine and R‐apomorphine) in two experimental conditions, with and without calcium in the bathing solution, and compared these effects with those obtained with the calcium antagonists verapamil and diltiazem. The present study shows that the alkaloids relax the uterine muscle but with different mechanisms of action. In Ca2+‐containing solution all three alkaloids relaxed the uterus previously contracted by KCl or acetylcholine, but in Ca2+‐free medium only R‐apomorphine was able to relax oxytocin‐induced contraction. The calcium antagonists, verapamil and diltiazem, relaxed KCl‐ or acetylcholine‐induced contraction in Ca2+‐containing solution, whereas they only relaxed oxytocin‐induced contraction in Ca2+‐free medium at much higher doses. These results suggest that glaucine and boldine behave as specific calcium entry blockers without affecting the contractile machinery or intracellular Ca2+ levels as apomorphine does. The absolute configuration (S‐glaucine and S‐boldine vs R‐apomorphine) may account for this different action.