M.A. Noguera

Different and common intracellular calcium-stores mobilized by noradrenaline and caffeine in vascular smooth muscle

SummaryNoradrenaline (NA) 1 μM and caffeine (CAF) 10 mM induce a contractile response in isolated rat aorta maintained at 37°C either in the presence or absence of extracellular calcium. In Ca-free media the contractile response was reduced and contractile activity of CAF only occurred at 25°C.NA induced a biphasic response in Ca-free medium, with a fast phasic contraction followed by a smaller more sustained contraction. The response induced by CAF consisted of a fast transient contraction which returned to a level below the resting tone. After washing, further addition of NA or CAF evoked no increase in smooth muscle tension. The influence of Mg-depletion in the extracellular medium on the contractile responses induced by NA and CAF in Ca-free medium was determined: similar response to NA or CAF in media with or without Mg after 2 min loading were observed, but after 15 min loading in Mg, Ca-free solution, the responses to NA or CAF were significantly higher than after incubation in Ca-free medium containing Mg. Differences were observed when contractile response to NA was elicited after the refilling process by loading the aortic strip in Ca-containing Mg-free solution. In this case, instead of recovering the magnitude of contraction there was a significant decrease. The existence of two independent intracellular Ca-pools releasable by NA, one of them also sensitive to CAF, is postulated. The refill of the Ca-store specific to NA is dependent on the presence of Mg in the extracellular medium. In contrast, the refill of the common Ca-pool releasable by NA and CAF is independent of the extracellular Mg, but its spontaneous emptying is temperature-, and Mg-dependent. This suggests the intermediacy of an enzymatic process to extrude the Ca-content of this store to the extracellular space.

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.

A possible structural determinant of selectivity of boldine and derivatives for the α1A-adrenoceptor subtype

1 The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9‐O‐methylboldine) and glaucine (2,9‐O‐dimethylboldine) on α1‐adrenoceptor subtypes was studied by examining [3H]‐prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective α1A‐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 α1A‐adrenoceptors was boldine (70 fold α1A‐selective) = predicentrine (60 fold, α1A‐selective) > glaucine (15 fold, α1A‐selective). 4 Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 μm) for 30 min at 37°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 selectivity of action for the a α1A‐adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2‐hydroxy function, induces a significant increase in α1A‐subtype selectivity and affinity.

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 β-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 β-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 β-arrestin 2 mediated internalization.

Vasodilator Effects of Liriodenine and Norushinsunine, Two Aporphine Alkaloids Isolated from Annona cherimolia, in Rat Aorta

The effect of two aporphines, liriodenine and norushinsunine, isolated from Annona cherimolia, were studied in the rat aorta in order to examine their mechanism of action. Both alkaloids (10(-7) - 10(-4) mol/l) showed relaxant effects on the contractions elicited by 10(-6) mol/l noradrenaline (NA) or 80 mmol/1 KCl, but, while liriodenine showed a nonspecific relaxant action on both spasmogens, norushinsunine was more potent on KCl-induced contraction. In Ca2+ free medium, both alkaloids (0.1 mmol/l) inhibited the responses elicited by NA, but not those elicited by caffeine. This inhibitory action occurred when the alkaloids were present during the release of the Ca2+ internal stores or during the refilling process. These results suggest that the two aporphines show a relaxant action in rat aorta which is mediated by an interaction with alpha1-adrenoceptors and an alteration of the Ca2+ entry via voltage-operated channels. Norushinsunine exhibits a certain degree of selectivity as an L-type Ca2+ channel blocker.

Relationships between structure and vascular activity in a series of benzylisoquinolines

1 1In 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 KC1, and a determination of the affinity of the compounds for α1‐adrenoceptors and calcium channel binding sites, with [#H]‐prazosin, [#H]‐nitrendipine and [#H]‐(+)‐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 2The three papaverine derivatives show greater affinity than papaverine for the [#H]‐prazosin binding site. They are therefore more selective as inhibitors of [#H]‐prazosin binding as opposed to [#H]‐(+)‐cis‐diltiazem, while papaverine appears to have approximately equal affinity for both. [#H]‐nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 μM. 3‐Methylisoquinoline had no effect on any of the binding sites assayed. 3 3Contractions evoked by noradrenaline (1 μM) 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 μM) 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 4Inositol phosphates formation induced by noradrenaline (1 μM) in rat aorta was inhibited by tetrahydropapaverine (100 μM) and tetrahydropapaveroline (300 μM), thus suggesting that α1D‐adrenoceptors are coupled to phosphoinositide metabolism in rat aorta. 5 5Unlike 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 6These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for α1‐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 α1‐adrenoceptor site and facilitates it with the Ca2+‐channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to α1‐adrenoceptors.

Intervention of two voltage-dependent calcium-entry pathways in the contractile response to acetylcholine and KCl in rat uterus

The contractile response of rat uterine smooth muscle was investigated. Verapamil and diltiazem concentration-dependently relax the sustained contractions induced by KCl (56 mmol/l) or acetylcholine (10(-4) mol/l). This inhibitory effect was not not freely reversed by washing the tissue and subsequently no contractile response was obtained in depolarized tissue, but a lower biphasic response (phasic and tonic) to acetylcholine was observed. Addition of cumulative concentrations of CaCl2 (1.2-19.2 mmol/l) induced a partial recovery of the contractile response to acetylcholine or KCl, but addition of MgCl2 (1.2-19.2 mmol/l did not. When the channel was reactivated by a third addition of KCl or acetylcholine after treatment with Ca2+, both spasmogens-induced phasic contractions recovered towards the initial configuration but the tonic component was not restored under any conditions. No recovery of mechanical response could be observed after Mg2+ treatment.

Relaxant activity of three aporphine alkaloids from Annona cherimolia on isolated aorta of rat

In the present study we tested the relaxant effect of three aporphine alkaloids—roemerine, anonaine and dehydroroemerine—isolated from the roots of Annona cherimolia, on isolated strips of rat thoracic aorta.

Actions of alpha-adrenoceptor blocking agents on two types of intracellular calcium stores mobilized by noradrenaline in rat aorta

SummaryIn isolated rat aortic strips noradrenaline induces a biphasic contractile response in Ca-free medium, associated with two different intracellular calcium pools, one of which is common to caffeine. We analyzed the mechanisms involved in the depletion and repletion of both intracellular Ca pools sensitive to noradrenaline in different experimental procedures in presence of prazosin, phentolamine and yohimbine.At 37°C the α-adrenergic blocking agents inhibited contractile responses to noradrenaline in Ca-free medium, with prazosin being highly selective. α2-adrenoceptors probably do not participate in the release of Ca from internal stores, as no contractile response was observed after addition of clonidine in Ca-free medium. This indicates that noradrenaline-induced Ca-release from internal stores is mainly due to activation of α1-adrenoceptors. At 25°C, these compounds failed to inhibit caffeine-induced contraction in Ca-free medium, but abolished the release of Ca from an intracellular store only sensitive to noradrenaline. This effect is attributale to a blockade of α1-adrenoceptors and/or inhibition of receptor-mediated signal transduction.

Differences in the Signaling Pathways of α1A- and α1B-Adrenoceptors Are Related to Different Endosomal Targeting

Aims To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin