M. Antonia Noguera

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.

Evidence that depletion of internal calcium stores sensitive to noradrenaline elicits a contractile response dependent on extracellular calcium in rat aorta

1 Noradrenaline 1 μm induced a contractile response in rat isolated aorta in the presence or in the absence of extracellular Ca2+ with depletion of intracellular Ca2+ stores. Thereafter, during incubation in the presence of Ca2+, an increase in the resting tone was observed. Such a contractile response did not occur after exposure to caffeine or 5‐hydroxytryptamine. 2 This increase in tension was inhibited in a concentration‐dependent manner by α‐adrenoceptor antagonists (prazosin, phentolamine and yohimbine), the non‐specific relaxing compound, papaverine and by the Ca2+‐entry blocker, nifedipine. Therefore, this contractile process is related to depletion of Ca2+ stores sensitive to noradrenaline and is linked to Ca2+ entry through voltage‐operated Ca2+ channels and α‐adrenoceptors. 3 Phentolamine and yohimbine did not block the Ca2+ refill pathway; prazosin and nifedipine inhibited the reuptake of Ca2+ by an internal store sensitive only to noradrenaline; papaverine inhibited the refilling of caffeine‐ and noradrenaline‐sensitive Ca2+‐stores.

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.

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.

Functional characterization of α1‐adrenoceptor subtypes in vascular tissues using different experimental approaches:a comparative study

The α1‐adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5‐methylurapidil (α1A selective) and BMY 7378 (α1D selective) have been used in different experimental procedures. Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC50‐contraction=7.32±0.04; pEC50‐IPs=6.03±0.08) than in the tail artery (pEC50‐contraction=5.71±0.07; pEC50‐IPs=5.51±0.10). Although the maximum contraction was similar in both tissues (Emax‐tail=619.1±55.6 mg; Emax‐aorta‐698.2±40.8 mg), there were marked differences in the ability of these tissues to generate intracellular second messengers the tail artery being more efficient (Emax‐tail=1060±147%; Emax‐aorta=108.1±16.9%). Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline‐induced maximal response in Ca2+‐containing or Ca2+‐free medium have been performed. A comparative analysis of the different procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed. The CICa was the simplest method to characterize the predominant α1‐adrenoceptor subtype involved in the functional response of a tissue. In aorta, where constitutively active α1D‐adrenoeptors are present, the use of different experimental procedures evidenced a complex equilibrium between α1D‐ and α1A‐adrenoceptor subtypes. The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the different experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

Modulatory Role of Magnesium on the Contractile Response of Rat Aorta to Several Agonists in Normal and Calcium‐free Medium

Abstract— Acute withdrawal of external Mg2+ increased basal tone of rat isolated aorta incubated in the presence of Ca2+. Above normal levels of Mg2+ (1–4 Mm) inhibited basal tone while much higher levels of the divalent cation (64–256 Mm) evoked contractile responses regardless of the presence of Ca2+. Contractile responses to noradrenaline (1μm) and KCl (80 Mm) were inhibited by addition of cumulative concentrations of Mg2+. Acetylcholine‐induced contractions in the presence of physiological concentrations of Mg2+ (1 Mm) decreased gradually to the basal tone, but a sustained contraction was observed in the absence of this ion. In Ca2+‐free medium, acetylcholine‐induced phasic responses indicate the existence of an acetylcholine‐sensitive Ca2+ store. KCl induced contraction only in Krebs solution, although a small residual contraction could be observed in Ca2+‐free medium in some experiments. Mg2+‐depletion in the extracellular medium increased contractile responses induced by acetylcholine and KCl in Ca2+‐free medium. These results suggest that extracellular Mg2+ modulates basal tone, Ca2+ channels and responsiveness to various agents in the absence of Ca2+.