Beyond lectures and practical courses: Teaching pharmacology using imaginative pedagogical tools.

Pharmacology has broadened its scope considerably in recent decades. Initially, it was of interest to chemists, doctors and pharmacists. In recent years, however, it has been incorporated into the teaching of biologists, molecular biologists, biotechnologists, chemical engineers and many health professionals, among others. Traditional teaching methods, such as lectures or laboratory work, have been superseded by the use of new pedagogical approaches to enable a better conceptualization and understanding of the discipline. In this article, we present several new methods that have been used in Spanish universities. Firstly, we describe a teaching network that has allowed the sharing of pedagogical innovations in Spanish universities. A European experience to improve prescribing safety is described in detail. The use of popular films and medical TV series in biomedical students shows how these audiovisual resources can be helpful in teaching pharmacology. The use of virtual worlds is detailed to introduce this new approach to teaching. The increasingly important area of the social aspects of pharmacology is also considered in two sections, one devoted to social pharmacology and the other to the use of learning based on social services to improve understanding of this important area. Finally, the use of Objective Structured Clinical Evaluation in pharmacology allows to know how this approach can help to better evaluate clinical pharmacology students. In conclusion, this article allows to know new pedagogical methods resources used in some Spanish universities that may help to improve the teaching of pharmacology.

Papel de la antitrombina III en cirugía cardiaca / Role of antithrombin III in cardiac surgery

La coagulación de la sangre suscita un interés multidisciplinar incrementado. La cirugía cardiaca produce cambios importantes en el delicado equilibrio entre los factores séricos pro- y anticoagulantes. El papel de la antitrombina iii se ha analizado después de conocer evidencias que relacionan bajos niveles de actividad de la proteína con la morbimortalidad postoperatoria. El aporte exógeno de antitrombina se considera con objeto de optimizar los resultados posquirúrgicos. Sus propiedades anticoagulante y antiinflamatoria intrínsecas despiertan un creciente interés y sugieren nuevas líneas de investigación (AU)

Coagulation of blood is of multidisciplinary interest. Cardiac surgery produces major changes in the delicate balance between pro-and anti-coagulant serum factors. The role of antithrombin iii has been analysed after finding evidence that associated decreased levels of protein activity to postoperative morbidity and mortality. Supplementing exogenous antithrombin is considered with the aim of optimising outcomes. Its intrinsic anticoagulant and anti-inflammatory properties have stimulated a growing interest, and suggests new lines of research (AU)

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.

Abordaje del problema de la hipertensión desde una oficina de farmacia: detección, educación sanitaria o seguimiento farmacoterapéutico. ¿Qué actuación muestra mayor eficiencia? / Approaching the problem of hypertension from a pharmacy: detection, health education and monitoring. Which activity shows more efficiency?

Introducción: La prevalencia de la hipertensión arterial en España aumenta con los años. Nos proponemos evaluar la función del farmacéutico desde la farmacia comunitaria como instrumento muy útil para mejorar el control y el manejo del paciente hipertenso, evaluando y comparando la eficiencia de las diferentes intervenciones que puede realizar para beneficiar al paciente hipertenso: detección de cifras altas de presión arterial (PA), educación sanitaria y seguimiento farmacoterapéutico. Método: Estudio observacional, prospectivo y de intervención, llevado a cabo en una farmacia de Valencia. Se ofertaba la toma de la PA a los participantes y se les pasaba una encuesta. Los pacientes eran asignados de forma aleatoria a un tipo de intervención: consejo farmacéutico, educación sanitaria o seguimiento farmacoterapéutico. A los 6 meses se evaluó la intervención. Resultados: Se propone la inclusión en el estudio a 220 pacientes, y aceptan participar 207, que se clasifican en 65 normotensos (31,4%), 64 prehipertensos (30,9%) y 78 hipertensos (37,7%). De los hipertensos, el 40% desconocía sus cifras elevadas de PA, y remitimos a los pacientes al médico. Se realizó un seguimiento farmacoterapéutico durante 6 meses en el grupo de pacientes con cifras de PA >140/90 mmHg, observándose diferencias en función de la actuación farmacéutica realizada. Conclusiones: El farmacéutico comunitario puede ser un elemento clave para conseguir mejorar la salud cardiovascular. Los programas de cribado de pacientes hipertensos parecen ser más coste-efectivos que el seguimiento semanal del paciente hipertenso(AU)

Introduction: The prevalence of hypertension in Spain is increasing over the years. In this study we evaluated the role of the community pharmacist as useful tool to improve the control and management of hypertensive patients. We evaluated and compared the effectiveness of three different interventions which could be developed in the community pharmacy to benefit hypertensive patients: detection of high blood pressure, health education and pharmaceutical care. Method: A prospective observational and intervention study was carried out in a community pharmacy from Valencia (Spain). Blood pressure determination was offered to adult people entering to the pharmacy. They answered a survey and the pharmacist acted differently depending on the day of the week: counseling, sanitary education and pharmaceuthical care, at 6 months, the pharmacist intervention were evaluated. Results: The study was proposed to 220 patients and 207 accepted. They are classified as normotensive (31.4%), prehypertensive (30.9%) and hypertensive (37.7%) depending on their arterial pressure. 40% of hypertensive patients, ignore their high levels blood pressure. In these cases, patients were referred to the doctor. In the group of patients with blood pressure above 140/90 mmHg, patients were followed for 6 months. Differences observed depending on the pharmacist actuation. Conclusions: The community pharmacist can be a key element in achieving better results in cardiovascular health. The implementation of screening programs in hypertensive patients appears to be more cost/effective than weekly monitoring of hypertensive patients(AU)

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.

[Role of antithrombin iii in cardiac surgery]. / Papel de la antitrombina iii en cirugía cardiaca.

Coagulation of blood is of multidisciplinary interest. Cardiac surgery produces major changes in the delicate balance between pro-and anti-coagulant serum factors. The role of antithrombin iii has been analysed after finding evidence that associated decreased levels of protein activity to postoperative morbidity and mortality. Supplementing exogenous antithrombin is considered with the aim of optimising outcomes. Its intrinsic anticoagulant and anti-inflammatory properties have stimulated a growing interest, and suggests new lines of research.

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion.

BACKGROUND AND PURPOSE: Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1. EXPERIMENTAL APPROACH: SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O(2) (•-) ) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively. KEY RESULTS: I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ET(B) mRNA expression, and down-regulated ET(A) mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ET(B) receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ET(B) receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ET(B) receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, N(ω) -nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O(2) (•-) production by increasing NADPH oxidase 1 (NOX-1) and p(47phox) mRNA. CONCLUSIONS AND IMPLICATIONS: Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ET(B) receptors and decrease in NO bioavailability.

Correlation between beta-adrenoceptors and G-protein-coupled receptor kinases in pretransplantation heart failure.

INTRODUCTION: Prolonged catecholamine overstimulation of the myocardium in chronic heart failure causes a reduction in the number and functionality of beta1-adrenoceptors (beta1-AR) of the heart. Desensitization of beta1-AR is mediated by their phosphorylation by a group of cytosolic kinases (G-protein-coupled receptor kinases GRK). In advanced heart failure, an increase in GRK levels associated with the severity of the disease has been observed. OBJECTIVE: The objective of this study was to analyze messenger RNA (mRNA) levels of beta1-AR in the myocardium of patients who underwent transplantation for advanced heart failure and their correlation with expression of the major cardiac isoenzymes of GRK. MATERIALS AND METHODS: Myocardial tissue samples were obtained from the left ventricles of 14 explanted hearts of patients who underwent transplantation for dilated (n = 7) and ischemic (n = 7) cardiomyopathy. RT-PCR techniques were used to analyze mRNA levels of beta1-AR and the isoenzymes GRK2, GRK3, and GRK5. RESULTS: We observed a significant correlation between beta1-AR and the 3 subtypes of GRK (R(2) = 0.668, 0.71, and 0.318, respectively). CONCLUSIONS: In patients with advanced heart failure pretransplantation, we observed a significant correlation between beta1-AR and GRK2 and GRK3 levels. GRK5, the subtype predominantly expressed in the myocardium, showed a lesser correlation with beta1-AR levels.

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.

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.

Mechanism of vascular relaxation by thaligrisine: functional and binding assays.

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.

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.

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.

Effect of divalent cations on the contractile response of rat aorta to depolarization before and after nifedipine treatment.

The influence of the divalent cations, Ca2+, Mg2+ and Ba2+, on the contractile response of the rat aorta to KCl and on the recovery of this response after nifedipine treatment was analyzed. KCl (80 mmol/l) promoted a two-phase (phasic and tonic) contractile response in Krebs solution but, as expected, no contractile response in Ca(2+)-free medium. In Mg(2+)-free medium, the phasic response to KCl was unaffected but the tonic one decreased slowly, suggesting that a long incubation time in the absence of Mg2+ (65 min) promotes a loss of or a change in the intracellular distribution of this ion that modifies Ca2+ entry through L channels or Ca2+ handling. Ba2+ (1.8 mmol/l) contracted the rat aorta in the absence or presence of Ca2+ but, when Ca2+ was not present, Ba2+ modified the contractile process so that a new addition of KCl did not reproduce the contractile response. After nifedipine treatment, no phasic response to KCl was observed and only a slow response appeared that increased as long as the KCl was present (45 min). After washing, KCl induced a phasic response similar to the standard response, but a smaller tonic one. Divalent cations accelerated the recovery of the channels involved in the tonic contraction. However, the recovery of the Ca2+ channels related to the phasic contraction was independent of divalent cations.