Jean-Claude Stoclet

Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta: A study with selective inhibitors

Three isoforms of cyclic nucleotide phosphodiesterase (PDE) have been recently isolated from aortic tissue and two of them specifically hydrolyzed adenosine 3,5-cyclic monophosphate (cAMP) and guanosine 3:5-cyclic monophosphate (cGMP), respectively (Lugnier et al., Biochem. Pharmac. 35, 1743, 1986). The role of these forms in controlling cyclic nucleotide levels and smooth muscle tone was investigated by the use of PDE inhibitors. The effects of selective inhibitors of the two forms specifically hydrolyzing cAMP or cGMP (cAMP-PDE and cGMP-PDE, respectively) were compared to those of non-selective inhibitors of the three aortic PDE forms, including the calmodulin-sensitive one (CaM-PDE). Relaxation responses and accumulation of tissue cAMP and cGMP induced by these drugs were studied in precontracted rat isolated aorta, and compared to the effects of isoprenaline and forskolin (stimulants of adenylate cyclase) or sodium nitroprusside (SNP) and sodium azide (stimulants of guanylate cyclase). The eight PDE inhibitors tested all relaxed aorta with potencies that correlated with their potencies as inhibitors of cAMP-PDE, but not of cGMP-PDE. At a concentration producing half-maximal relaxation, all PDE inhibitors induced a moderate but significant accumulation of cAMP, which was comparable to the accumulation of cAMP elicited by half-maximally relaxing concentrations of adenylate cyclase stimulating agents. At this concentration, some PDE inhibitors (M&B 22,948, dipyridamole and to a lesser extent, trequinsin) also induced a significant increase in cGMP levels, of the same order of magnitude as that caused by agents stimulating guanylate cyclase. However, the cGMP-increasing effect of these inhibitors was dissociated from their relaxing effect. In particular, the relaxing concentrations of M&B 22,948 (a selective inhibitor of cGMP-PDE) were clearly higher than the cGMP-increasing concentrations of the compound. At a concentration at which they elicited 10% relaxation by themselves, the selective cAMP-PDE inhibitor, rolipram, as well as the mixed inhibitor of cAMP- and cGMP-PDE, AAL 05 (a cilostamide analogue) enhanced both the cAMP-increasing and the relaxing effect of isoprenaline. Under the same conditions, no clear enhancement of the relaxation induced by SNP was observed. Only M&B 22,948 showed a slight potentiating effect on SNP-induced relaxation, but this effect was limited to low concentrations of SNP (less than 10 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

Endothelial mediated inhibition of contraction and increase in cyclic GMP levels evoked by the α‐adrenoceptor agonist B‐HT 920 in rat isolated aorta

1 In the presence of endothelium maximal contractions of rat aorta preparations evoked by B‐HT 920 were about 10% of those evoked in the absence of endothelium. 2 6‐Allyl‐2‐amino‐5,6,7,8‐tetrahydro‐4H‐thiazolo‐(4,5‐d)azepin dihydrochloride (B‐HT 920, 0.1 μM to 0.1 mM) induced concentration‐dependent contractions of rat aorta in the absence of endothelium. Maximal contractions were comparable in magnitude to those induced by noradrenaline. 3 In the presence of endothelium but not in its absence B‐HT 920 (0.1 mM) stimulated an increase in tissue cyclic GMP levels of about 2 fold. Levels of cyclic AMP were unaffected. Removal of endothelium reduced basal tissue levels of cyclic GMP. 4 The guanylate cyclase inhibitor methylene blue (0.5 JIM) potentiated B‐HT 920‐induced contractions in the presence of endothelium and inhibited increases in cyclic GMP. 5 In the presence of endothelium 5,8,11,14‐eicosatetraynoic acid (ETYA; 0.1 mM), an inhibitor of both lipoxygenase and cyclo‐oxygenase systems, inhibited the B‐HT 920‐induced increase in cyclic GMP but did not potentiate B‐HT 920‐induced contractions. ETYA also antagonized B‐HT 920‐induced contractions in the absence of endothelium. 6 It is concluded that endothelium continuously releases a product or products which influence the smooth muscle. Inhibition of B‐HT 920‐induced contractions in the presence of endothelium is associated with increased tissue levels of cyclic GMP.

Insensitivity of calcium‐dependent endothelial stimulation in rat isolated aorta to the calcium entry blocker, flunarizine

1 In rat aortic segments complete with endothelium, acetylcholine (1 μM) relaxed noradrenaline, phenylephrine and prostaglandin F2α (PGF2α‐induced contractions of various magnitudes. Maximal 1 μM phenylephrine‐induced contractions were relaxed to a greater extent than were maximal contractions induced by the other two agonists. 2 Contractions elicited by various concentrations of phenylephrine and PGF2α in the presence of a maximal effective concentration of the calcium entry blocker flunarizine (3 μM) were relaxed by acetylcholine to about the same residual tension as were contractions elicited in the absence of flunarizine. 3 Acetylcholine (1 μM) and phenylephrine (1 μM) increased tissue levels of guanosine cyclic 3′5′‐monophosphate (cyclic GMP) by about 37 fold and 2 fold respectively. Preincubation of tissues in the absence of calcium abolished these agonist‐induced increases in cyclic GMP levels, but preincubation with flunarizine had no significant effect on the increase in cyclic GMP level induced by the agonists. Pretreatment with flunarizine had no significant effect on the basal tissue level of cyclic GMP, but pretreatment in calcium‐free solution reduced the basal tissue level of the cyclic nucleotide by about half. 4 It is concluded that in rat aorta, endothelium‐dependent acetylcholine‐induced relaxation and endothelium‐dependent acetylcholine and phenylephrine‐induced increases in tissue levels of cyclic GMP, are dependent on extracellular calcium, but are not antagonized by flunarizine. This may indicate that if calcium channels of endothelial cells are activated by these agonists, their characteristics are not identical with those of the calcium channels of the smooth muscle cells.

Tissue and substrate specificity of inhibition by alkoxy-aryl-lactams of platelet and arterial smooth muscle cyclic nucleotide phosphodiesterases relationship to pharmacological activity

Alkoxy-aryl-lactams (cilostamide, AAL 05, ZK 62 711, Ro 20-1724) inhibit differently cAMP or cGMP phosphodiesterases from blood platelets or vascular smooth muscle. Cilostamide (IC50 0.23 microM) and AAL 05 (IC50 0.15 microM) are 100 times more potent towards platelet cAMP phosphodiesterase whereas ZK 62 711 (IC50 2 microM) and Ro 20-1724 (IC50 33 microM) inhibit more selectively the enzyme from aorta. The substrate specificity of the inhibitors is different in the two tissues: ZK 62 711 and cilostamide are respectively 345 and 290 times more potent as inhibitor of cAMP than cGMP phosphodiesterase from vascular smooth muscle (ZK 62 711) or platelets (cilostamide). M + B 22,948 selectively inhibits cGMP phosphodiesterase with an IC50 of 9 or 24 microM on platelet or aorta enzyme, respectively. In general, the potencies of spasmolytic and platelet inhibitor effects vary from one drug to the other with the potency of inhibition of phosphodiesterase from the corresponding tissue. These data suggest that phosphodiesterases from platelets are different from those of arterial smooth muscle.

Effect of vasoactive intestinal polypeptide (VIP) on cyclic AMP level and relaxation in rat isolated aorta

The effects of vasoactive intestinal polypeptide (VIP) on cyclic nucleotide (cAMP and cGMP) levels and smooth muscle relaxation were investigated in rat isolated aorta and compared to those of the beta-adrenergic agonist isoprenaline. VIP increased the cAMP level of rat aorta in a concentration-dependent manner with an EC50 of 0.1 microM. VIP 1 microM maximally increased the cAMP level 7 fold, whereas the beta-adrenergic agonist isoprenaline 10 microM elevated the cAMP level only 2.5 fold. VIP 1 microM relaxed the precontracted rat aorta by only about 16% whereas isoprenaline 10 microM induced a relaxation of about 86%. VIP did not alter the cGMP level and its effect on cAMP content was not changed in the presence of indomethacin (5 microM). No quantitative correlation could thus be established between increases in total tissue levels of cAMP and the degree of relaxation in rat isolated aorta.

Dissociation between endothelium-mediated increases in tissue cGMP levels and modulation of aortic contractile responses

SummaryBoth methoxamine and clonidine elicited similar maximal contractions of rat isolated aorta in the absence of endothelium. These contractions were not associated with changes in tissue levels of cGMP or cAMP. In the presence of endothelium maximal methoxamine-induced contractions were not less than those elicited in the absence of endothelium but maximal clonidine-induced contractions were reduced to about 10% of those in the absence of endothelium. However, in the presence of endothelium both methoxamine and clonidine induced similar increases in tissue cGMP levels of about 1.5 to 2 fold; cAMP levels were unchanged. There is therefore a dissociation between endothelium-mediated inhibition of maximal contractile responses and increases in tissue levels of cGMP.

Characterization of two distinct alpha-adrenoceptor binding sites in smooth muscle cell membranes from rat and bovine aorta

SummaryIn order to characterize postjunctional alpha adrenoceptor binding sites of aortic smooth muscle, the specific binding of (3H)prazosin and (3H)yohimbine to membranes prepared from the medial layers of rat and bovine thoracic aorta was investigated. Binding of (125I)-BE 2254 (2-[B-(4-hydroxyphenyl)-ethylaminomethyl] tetralone) and (3H)RX 781094 (idazoxan) was also examined in bovine membranes. Each of the ligands displayed saturable, specific binding to a single population of sites; the KD values of the respective ligands were similar in the two animal species. The number of (3H)prazison and (125I)BE 2254 binding sites (160–190 fmol · mg protein−1 in the two species) was higher than the number of (3H)yohimbine and (3H)RX 781094 binding sites (110–120 fmol · mg protein−1 in the bovine and 50 fmol · mg protein−1 in the rat). Alpha-adrenoceptor ligands inhibited binding of the ligands with the following orders of potency: prazosin > BE 2254 > yohimbine > RX 781094 > clonidine in the case of (3H)-prazosin, and yohimbine > RX 781094 > clonidine > prazosin in the case of (3H)yohimbine. Methoxamine, in concentrations up to 10 μM, was without effect on the binding of either ligand. The absence or presence of Na+, K+ or Ca2+ added at physiological concentrations did not change the order of potency of displacing ligands whereas Ca2+ reduced by 50% the numbers of (3H)prazosin and (3H)-yohimbine sites and Na+ increased by 3-fold the affinity of (3H)yohimbine.It is concluded that post-junctional membranes from rat and bovine aortic smooth muscle contain two distinct α1- and α2-adrenoceptor binding sites, the number of the latter being less than the number of the former.

Modulation by endothelium of contractile responses in rat aorta in absence and presence of flunarizine

1 The possible modulation by endothelium of phenylephreine‐ and prostaglandin F2α‐induced mobilization of calcium for contraction in the rat aorta has been investigated. Contractions elicited by these and other agonists are inhibited in the presence of endothelium. 2 For any single concentration of phenylephrine in the presence of endothelium, the initial phasic components of contractions were significantly greater, the maximal contractions were achieved sooner and were less well maintained as compared to contractions elicited in the absence of endothelium. 3 The kinetic characteristics of contractions stimulated by single concentrations of PGF2α were similar in the presence and absence of endothelium and did not exhibit initial phasic components of contraction. 4 Sub‐maximal contractions elicited by both PGF2α and phenylephrine in the absence of endothelium were inhibited to a greater extent by flunarizine 3 μM than equieffective contractions elicited in the presence of endothelium. 5 Maximal contractions elicited by phenylephrine (1 μM) were inhibited to a similar extent by flunarizine in the presence and absence of endothelium, but maximal contractions elicited by PGF2α (30 μM) were inhibited by flunarizine to a greater extent in the presence than in the absence of endothelium. 6 It is concluded that an endothelium‐derived factor, perhaps distinct from endothelium‐derived relaxing factor, can modulate the ability of both phenylephrine and PGF2α to mobilize calcium for contraction. This modulatory effect is associated with an enhanced mobilization of intracellular calcium. Thus, submaximal concentrations of both agonists were less dependent on extracellular calcium than on intracellular calcium to elicit contractions in the presence of endothelium, as compared to contractions elicited in the absence of endothelium.

α-adrenoceptor antagonistic and calcium antagonistic effects of nicergoline in the rat isolated aorta

The activity of the alpha-adrenoceptor antagonist nicergoline, a molecule composed of two constituent parts, ergoline and bromonicotinic acid, was investigated in the rat isolated aorta. Nicergoline (10 nM-0.1 microM) displaced concentration-effect curves elicited by noradrenaline and phenylephrine to the right and inhibited maximal responses elicited by both alpha-adrenoceptor agonists without significantly affecting prostaglandin F2 alpha-induced contractions. Higher concentrations of nicergoline (1 microM-50 microM) displaced to the right the concentration-effect curves elicited by calcium in a depolarizing medium. This calcium antagonist activity was not shared by either of the constituent parts. Nicergoline 100 microM abolished the 45Ca influx induced into rat aorta by 100 mM K+-containing physiological solution. The selectivity of nicergoline for alpha 1-adrenoceptors seen in binding experiments also depends on the presence of the bromonicotinic moiety of the molecule. It is concluded that nicergoline, but not its substituent parts, displays both alpha 1-adrenoceptor and calcium antagonism. The latter property may account for some of the observed effects of this compound.

Phorbol ester inhibition of vasopressin-induced calcium efflux from cultured rat aortic myocytes

The effect of the protein kinase C activator TPA was investigated on AVP‐induced 45Ca release from rat aortic myocytes. In the nanomolar range TPA, but not 4β‐phorbol, reduced the brief 45Ca efflux produced by AVP in the presence or in the absence of extracellular calcium. The maximal effect of TPA was to abolish the response to a half maximally active concentration of AVP, and to reduce by 50% the maximal response to the hormone. These results suggest that protein kinase C activation can exert a negative control on the early AVP‐induced calcium mobilization in vascular smooth muscle.