The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting ß-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by ß-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on ß-arrestin 2 mediated internalization.

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

BACKGROUND AND PURPOSE: To analyse the relative contribution of ß1 -, ß2 - and ß3 -adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways. EXPERIMENTAL APPROACH: Rat mesenteric resistance artery (MRA) and aorta were used to analyse the Adrb expression by real-time-PCR and immunohistochemistry, and for the pharmacological characterization of Adrb-mediated activity by wire myography and tissue nucleotide accumulation. KEY RESULTS: The mRNAs and protein for all Adrb were identified in endothelium and/or smooth muscle cells (SMCs) in both vessels. In MRA, Adrb1 signalled through cAMP, Adrb3 through both cAMP and cGMP, but Adrb2, did not activate nucleotide formation; isoprenaline relaxation was inhibited by propranolol (ß1 , ß2 ), CGP20712A (ß1 ), and SQ22536 (adenylyl cyclase inhibitor), but not by ICI118,551 (ß2 ), SR59230A (ß3 ), ODQ (soluble guanylyl cyclase inhibitor), L-NAME or endothelium removal. In aorta, Adrb1 signalled through cAMP, while ß2 - and ß3 -subtypes through cGMP; isoprenaline relaxation was inhibited by propranolol, ICI118,551, ODQ, L-NAME, and to a lesser extent, by endothelium removal. CL316243 (ß3 -agonist) relaxed aorta, but not MRA. CONCLUSION AND IMPLICATION: Despite all three Adrb subtypes being found in both vessels, Adrb1, located in SMCs and acting through the adenylyl cyclase/cAMP pathway, are primarily responsible for vasodilatation in MRA. However, Adrb-mediated vasodilatation in aorta is driven by endothelial Adrb2 and Adrb3, but also by the Adrb2 present in SMCs, and is coupled to the NO/cGMP pathway. These results could help to understand the different physiological roles played by Adrb signalling in regulating conductance and resistance vessels.

Anisakis simplex s.l.: Larvicidal activity of various monoterpenic derivatives of natural origin against L3 larvae in vitro and in vivo.

This paper describes the activity against Anisakis simplex s.l. L(3) larvae of six monoterpenic derivatives obtained from different essential oils, (alpha-pinene, beta-pinene, ocimene, myrcene, geranyl acetate, and cineole). In in vitro assays, alpha-pinene, ocimene and cineole showed high activity at a concentration of 125 microg/mL (48h) but only alpha-pinene and ocimene were active at 62.5 microg/mL. In in vivo assays, L(3) larvae and study compounds were simultaneously administered per os to Wistar rats. The most active compound was alpha-pinene, finding lesions in only 20% of treated rats versus 98% of controls. Further in vivo studies are required to investigate whether addition of these compounds to food could have a prophylactic effect, reducing the pathogenicity of A. simplex s.l. L(3) in humans, and to explore any possible synergy among compounds.

Acute and chronic captopril, but not prazosin or nifedipine, normalize alterations in adrenergic intracellular Ca2+ handling observed in the mesenteric arterial tree of spontaneously hypertensive rats.

The effect of hypertension and acute (36-h) or chronic (from age 6 to 16 weeks) antihypertensive treatment with prazosin (2 mg kg(-1) per day), nifedipine (50 mg kg(-1) per day), or captopril (50 mg kg(-1) per day) on Ca2+ mobilization due to alpha1-adrenoceptor activation was analyzed in functional studies using arterial rings [four conductance/distributing vessels: aorta, main mesenteric, iliac, and tail arteries and two resistance vessels; first and second small mesenteric artery branches obtained from spontaneously hypertensive rats (SHR, 6 and 16 weeks old) and age-matched Wistar Kyoto rats (WKY)]. Maximal response to noradrenaline in the presence of extracellular Ca2+ is not affected by hypertension or by the antihypertensive treatment. The extracellular Ca2+-independent contractile responses increased with age in iliac, tail, and small mesenteric arteries (SMA) and were further increased in SHR in SMA from both young and adult animals and in the main mesenteric artery of adult SHR. In main mesenteric artery, this increased contraction in SHR was associated with a higher increase in cytosolic [Ca2+] mobilized by noradrenaline without changes in the total stored Ca2+. Acute or chronic treatment with captopril abolished the differences observed between WKY and SHR in the noradrenaline-induced contraction in mesenteric arteries loaded in Ca2+-free medium. In contrast, animals acutely treated with prazosin or chronically treated with either prazosin or nifedipine exhibit the same differences in Ca2+ handling than untreated rats. In conclusion, these differences are not a consequence of increased blood pressure but precede it and can only be normalized by inhibition of the rennin-angiotensin system.

Vascular activity of (-)-anonaine, (-)-roemerine and (-)-pukateine, three natural 6a(R)-1,2-methylenedioxyaporphines with different affinities for alpha1-adrenoceptor subtypes.

We have studied the mechanism of action of three 6a( R)-1,2-methylenedioxyaporphines as vasorelaxant compounds. The alkaloids assayed showed different affinities for the three human cloned alpha (1)-adrenoceptor (AR) subtypes stably expressed in rat-1 fibroblasts, showing lower affinity for alpha(1B)-AR with regard to the alpha(1A)- or alpha(1D)-subtypes. These three natural compounds are more potent inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. As all these alkaloids inhibited noradrenaline (NA)-induced [ (3)H]-inositol phosphate formation in cerebral cortex and rat tail artery, they may be safely viewed as alpha (1)-AR antagonists, as is demonstrated by the vasorelaxant responses observed in isolated rat tail artery and/or aorta precontracted with NA. The alkaloids also inhibited the contractile response evoked by KCl (80 mM) but with a lower potency than that shown against NA-induced contraction. We have also examined their ability to inhibit the different forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aortic smooth muscle and endothelial cells, with negative results. We conclude that N-methylation favours the interaction of (R)-aporphines with all alpha (1)-AR subtypes, and that the topography of the binding site recognizing the basic or protonated nitrogen atom is similar in all three alpha (1)-AR subtypes. The presence of a hydroxy group at C-11 has different effects on the affinity for each alpha (1)-AR subtype but decreases the affinity for Ca (2+) channels. These results confirm and extend the view that subtle changes in the hydroxylation patterns on the aromatic ring of the aporphine structure affect the interactions of these compounds with the three alpha (1)-AR subtypes in different ways, suggesting that the binding site recognizing the aporphine skeleton is different in each of the three subtypes.

[Agenesis of the gallbladder]. / Agenesia de la vesícula biliar.

The agenesis of the gall bladder is a rare congenital abnormality that usually is diagnosed as cholecystolithiasis. Therefore it is an intraoperative discovery. In this work two new cases are added to casuistry; both them have received laparotomic treatment. As those cases were symptomatic patients, the most common clinical manifestations are described. We insist in tactics to discard ectopic locations and the different options for the intraoperative and postoperative study; this last situation seems to be the most used behavior on patients approached laparoscopically.

Agenesia de la vesícula biliar. / [Agenesis of the gallbladder]

The agenesis of the gall bladder is a rare congenital abnormality that usually is diagnosed as cholecystolithiasis. Therefore it is an intraoperative discovery. In this work two new cases are added to casuistry; both them have received laparotomic treatment. As those cases were symptomatic patients, the most common clinical manifestations are described. We insist in tactics to discard ectopic locations and the different options for the intraoperative and postoperative study; this last situation seems to be the most used behavior on patients approached laparoscopically.

Agenesia de la vesícula biliar / Agenesis of the gallbladder

The agenesis of the gall bladder is a rare congenital abnormality that usually is diagnosed as cholecystolithiasis. Therefore it is an intraoperative discovery. In this work two new cases are added to casuistry; both them have received laparotomic treatment. As those cases were symptomatic patients, the most common clinical manifestations are described. We insist in tactics to discard ectopic locations and the different options for the intraoperative and postoperative study; this last situation seems to be the most used behavior on patients approached laparoscopically. (AU)

Agenesia de la vesícula biliar / Agenesis of the gallbladder

The agenesis of the gall bladder is a rare congenital abnormality that usually is diagnosed as cholecystolithiasis. Therefore it is an intraoperative discovery. In this work two new cases are added to casuistry; both them have received laparotomic treatment. As those cases were symptomatic patients, the most common clinical manifestations are described. We insist in tactics to discard ectopic locations and the different options for the intraoperative and postoperative study; this last situation seems to be the most used behavior on patients approached laparoscopically.

Comparative effects of the novel vasotocin analogue F-180 vs. vasopressin and terlipressin on systemic and splanchnic isolated vessels from portal hypertensive rats.

F-180 has been proposed as a new vasopressin analogue for the treatment of portal hypertension. This study investigates the contractile profile of F-180 compared to vasopressin and its analogue terlipressin on isolated systemic and splanchnic vessels from sham-operated and partial portal vein ligated (PPVL) rats. F-180 (10(-9)-10(-6) M), vasopressin (10(-11)-10(-8) M) and terlipressin (10(-9)-10(-4) M) induced contraction of the mesenteric vein, aorta, iliac, tail and mesenteric arteries. The order of potency in these vessels was vasopressin (pD2 approximately 9) > F-180 (pD2 approximately 8) > terlipressin (pD2 approximately 6). Significant (P<0.01) differences between sham-operated and PPVL rats were noticed exclusively in the mesenteric vein, being the maximal effect of the three agonists at least twice greater in PPVL rats than in sham-operated rats. The order of sensitivity to the vasoconstrictors in vessels from PPVL rats was aorta < mesenteric artery << iliac artery approximately equal tail artery approximately equal mesenteric vein. The contractile profile of these peptides in each vessel from PPVL animals was quite similar, except in the mesenteric vein and the aorta. F-180 showed higher efficacy (P<0.01) than terlipressin in the mesenteric vein and lower (P<0.05) efficacy than vasopressin in the aorta. These findings suggest the existence of a vasoconstrictor territorial selectivity for vasopressin and its analogues, which could justify the efficacy of these drugs in portal hypertension therapy. In particular, F-180 appears to be a viable alternative to the classic vasopressin analogues.

Role of cyclic nucleotide phosphodiesterase isoenzymes in contractile responses of denuded rat aorta related to various Ca2+ sources.

We have examined the cyclic nucleotide phosphodiesterase isoforms (PDE) involved in the contractile response of rat aorta to different agonists and different experimental procedures for use in functional studies. The inhibitory effect of AAL 05 on the different PDEs isolated from bovine aortic smooth muscle was examined. Compound AAL 05 appeared to be a selective PDE3 inhibitor. We analyzed the ability of the non-selective inhibitor IBMX (3-isobutyl-1-methylxanthine) and the isoenzyme selective inhibitors nimodipine (type 1), AAL 05 (6-(N-methyl-N-cyclohexyl butyl carboxamide) quinolin-2-one) and SK&F 94120 (5-(4-acetamidophenyl) pyrazin-2(1H)-one; type3), rolipram (type4) and zaprinast (type5) to affect the contractile responses of denuded rat aortic rings to KCl (80 mM) and noradrenaline (NA, 1 microM) in the presence or absence of extracellular Ca2+. Rolipram (10-100 microM) and zaprinast (1-100 microM) failed to relax the aortic strips, but IBMX (0.1-30 microM), nimodipine (1 fM10 microM), AAL 05 (0.01-100 microM) and SK&F 94120 (0.1-100 microM) produced a concentration-dependent relaxation or inhibition of contractile responses to the different agonists, but the pIC50 obtained for each inhibitor was different depending on the experimental procedure. Except for nimodipine (a Ca2+ channel blocker), all the PDE inhibitors showed the following rank of potency: pIC50 on NA-induced contractions in Ca2+-free medium > pIC50 on NA-induced contractions in Ca2+-containing solution > pIC50 on depolarizing solution-induced contraction. This ranking apparently depends on the differences in the Ca2+ sources. We obtained a good correlation between the pKi of PDE3 inhibitors in biochemical studies and the pIC50 on NA-induced contraction in Ca2+-free medium. In conclusion, PDE1 and PDE3 isoenzymes play an important role as modulators of rat aortic smooth muscle contractility regardless of the experimental procedure used. Since intracellular mechanisms are more dependent on PDE activity, experimental procedures performed in absence of extracellular calcium are the most suitable for analyzing the modulatory role of PDE inhibitors.

Functional evidence of a constitutively active population of alpha(1D)-adrenoceptors in rat aorta.

After depletion of intracellular calcium stores sensitive to noradrenaline, a spontaneous increase in the resting tone (IRT) when incubated in Ca(2+)-containing solution was observed in isolated rat aorta, but not in tail artery. This IRT does not depend on agonist activation of alpha(1)-adrenoceptors but it is inhibited by prazosin. A close relationship was found between the inhibitory potencies of prazosin (pIC(50) = 9.833), BMY 7378 (pIC(50) = 8.924), and 5-methylurapidil (pIC(50) = 7.883) against IRT and their affinities for cloned alpha(1D)-adrenoceptors. Chloroethylclonidine (100 micromol. l(-1)) did not inhibit the IRT. After depletion of internal calcium stores by noradrenaline in absence of the agonist, loading in Ca(2+)-containing solution also brings about an increase in the inositol phosphate (IP) levels in rat aorta (not seen in tail artery) that is inhibited by prazosin (1 micromol. l(-1)), BMY 7378 (10 micromol. l(-1)), and 5-methylurapidil (10 micromol. l(-1)), thus confirming the results obtained in contractile studies. Chloroethylclonidine (100 micromol. l(-1)) did not inhibit this IP accumulation. The fact that the IRT and the IP accumulation related to it can be selectively inhibited by different alpha(1)-adrenoceptor antagonists suggests the existence of a population of alpha(1D)-adrenoceptors that show constitutive activity in rat aorta, not in tail artery.

Effect of divalent cations on KCl- and noradrenaline-induced contractile responses in rat aorta after nifedipine treatment.

Nifedipine (1 microM) relaxed the sustained contractile responses induced by 1 microM noradrenaline or 60 mM KCl in rat aortic strips. After washing, a second addition of the spasmogens gave smaller tonic contractions than the first one. Even more, a third addition of KCl also gave a smaller contraction than the first one, but a complete recovery of the contractile response to noradrenaline was obtained by a third addition of this agonist. Application of cumulative amounts of Ca2+ or Ba2+ (2.4-24 mM) on the residual contraction in response to these agents after nifedipine treatment, but in the absence of the blocker, restored the magnitude of the contractile responses. Addition of cumulative amounts of Mg2+ (2.4-24 mM) did not modify or even relax the contractile responses to KCl and noradrenaline, respectively.

Halogenated derivatives of boldine with high selectivity for alpha1A-adrenoceptors in rat cerebral cortex.

The selectivity of 3-nitrosoboldine and different halogenated derivatives of boldine (3-bromoboldine, 3,8-dibromoboldine and 3-chloroboldine) for alpha1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. In the competition experiments [3H]-prazosin binding was inhibited completely by all the compounds tested. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components. The relative order of affinity and selectivity for alpha1A-adrenoceptors was 3-bromoboldine = 3,8-dibromoboldine = 3-chloroboldine > boldine > 3-nitrosoboldine. The competition curves for 3-bromoboldine remained shallow and biphasic following chloroethylclonidine treatment. Whereas the relative contribution of the high affinity sites increased, the 3-bromoboldine affinities at its high and low affinity sites remained similar to those obtained in untreated membranes. 3-Bromoboldine, 3,8-dibromoboldine, 3-chloroboldine and 3-nitrosoboldine did not significantly displace [3H]-(+)-cis-diltiazem binding to rat cerebral cortex membranes. This activity was lower than that shown by boldine. Compared to boldine, halogen (bromine or chlorine) substitution at position 3 increases the alpha1A-adrenoceptor subtype selectivity and decreases the affinity for the benzothiazepine binding site at the calcium channel. Further halogen substitution at position 8 did not significantly improve this activity with respect to 3-bromoboldine. In contrast, the NO substitution at position 3 of boldine (3-nitrosoboldine) gives a loss of affinity and selectivity for alpha1-adrenoceptor subtypes.

Characterization of two different Ca2+ entry pathways dependent on depletion of internal Ca2+ pools in rat aorta.

Ryanodine (10 microM), thapsigargin (1 microM) and cyclopiazonic acid (10 microM) 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 microM) 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 microM), thapsigargin (1 microM) and cyclopiazonic acid (10 microM). 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 microM) 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 alpha1-adrenoceptor-sensitive Ca2+ pool.

Relationships between structure and vascular activity in a series of benzylisoquinolines.

1. In the present work, the properties of 3-methyl isoquinoline, 3,4-dihydropapaverine, tetrahydropapaverine and tetrahydropapaveroline were compared with those of papaverine and laudanosine. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of the compounds for alpha1-adrenoceptors and calcium channel binding sites, with [3H]-prazosin, [3H]-nitrendipine and [3H]-(+)-cis-diltiazem binding to rat cerebral cortical membranes. The effects of papaverine derivatives on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. The three papaverine derivatives show greater affinity than papaverine for the [3H]-prazosin binding site. They are therefore more selective as inhibitors of [3H]-prazosin binding as opposed to [3H]-(+)-cis-diltiazem, while papaverine appears to have approximately equal affinity for both. [3H]-nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 microM. 3-Methylisoquinoline had no effect on any of the binding sites assayed. 3. Contractions evoked by noradrenaline (1 microM) in rat aorta were inhibited in a concentration-dependent manner by 3,4-dihydropapaverine, tetrahydropapaverine and with a lower potency, by tetrahydropapaveroline. In Ca2+-free solution, tetrahydropapaverine and to a lesser extent, tetrahydropapaveroline, inhibited the noradrenaline (1 microM) evoked contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (10 mM). This suggests that these alkaloids do not act at the intracellular level, unlike papaverine which inhibits the contractile response to caffeine and noradrenaline. 4. Inositol phosphates formation induced by noradrenaline (1 microM) in rat aorta was inhibited by tetrahydropapaverine (100 microM) and tetrahydropapaveroline (300 microM), thus suggesting that alpha1D-adrenoceptors are coupled to phosphoinositide metabolism in rat aorta. 5. Unlike papaverine, which has a significant effect on all the PDE isoforms, the three alkaloids assayed did not have an inhibitory effect on the different forms of PDE isolated from bovine aorta. 6. These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for alpha1-adrenoceptors and a lower affinity for benzothiazepine sites in the Ca2+-channel than papaverine. This structural feature also implies a loss of the inhibitory activity on PDE isoforms. The planarity of the isoquinoline ring (papaverine) impairs the interaction with the alpha1-adrenoceptor site and facilitates it with the Ca2+-channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to alpha1-adrenoceptors.

Capacitative Ca2+ entry associated with alpha1-adrenoceptors in rat aorta.

In rat aorta, depletion of internal Ca2+ stores by addition of noradrenaline (1 microM) 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(-11) M) 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 (10 microM). Cromakalim (10(-10)-10(-6) M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10 microM). 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 alpha 1-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 response evoked by noradrenaline (10 microM) in guinea-pig aorta (IC50 = 1.4 +/- 0.2 microM) while the KCl depolarizing solution (60 mM)- or the Ca2+ (1 mM)-induced contractions were only partially affected by boldine up to 300 microM. 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 alpha 1-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 alpha 1-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 Ca(2+)-containing Krebs solution was not affected by a 60 min incubation period with different doses of S-(+)-boldine (1-300 microM). Papaverine inhibited partially this caffeine-induced contraction at the maximal dose used (100 microM). 5. Inositol phosphates formation induced by noradrenaline (10 microM) in guinea-pig thoracic aorta was inhibited by S-(+)-boldine (30 microM) but not by papaverine (10 microM). 6. Contractions of guinea-pig trachea caused by acetylcholine (100 microM) or histamine (10 microM) were not modified by S-(+)-boldine (0.1-100 microM). 7. These results provide evidence that S-(+)-boldine, an aporphine alkaloid, has interesting properties as an alpha 1-adrenoceptor blocker in vascular smooth muscle, and acts as a competitive antagonist of the alpha 1-adrenoceptor present in the guinea pig aorta.

A possible structural determinant of selectivity of boldine and derivatives for the alpha 1A-adrenoceptor subtype.

1. The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-O-methylboldine) and glaucine (2,9-O-dimethylboldine) and alpha 1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective alpha 1A-adrenoceptor antagonists. 2. In the competition experiments [3H]-prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]-prazosin specific binding sites. 3. Boldine, predicentrine and glaucine also competed for [3H]-prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30-40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for alpha 1A-adrenoceptors was boldine (70 fold alpha 1A-selective) = predicentrine (60 fold, alpha 1A-selective) > glaucine (15 fold, alpha 1A-selective). 4. Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 microM) for 30 min at 37 degrees C followed by thorough washing out reduced specific [3H]-prazosin binding by approximately 70%. The CEC-insensitive [3H]-prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76). 5. These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectively of action for the alpha 1A-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in alpha 1A-subtype selectivity and affinity.

Functional evidence of inverse agonism in vascular smooth muscle.

1. In the present study, depletion of internal Ca2+ stores sensitive to noradrenaline (1 microM) 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 microM) reproduces the same processes as noradrenaline but clonidine (1 microM) does not, indicates that alpha(1)-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 (alpha(1A)- and alpha(1D)-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 microM) has no effect on this response, the participation of alpha(1B)-adrenoceptors can be ruled out. 4. Contractile responses to noradrenaline (1 microM) 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 alpha(1B)-adrenoceptor subtype.