Lacidipine: a dihydropyridine calcium antagonist with antioxidant activity.

Lacidipine, a new, long-acting antihypertensive dihydropyridine calcium antagonist was tested for potential antioxidant effect in a series of tests that consider specific radical species. A direct quenching of several radical species could be measured. Moreover, in biological membranes deriving from rat brain tissue, lacidipine showed an activity comparable to reference antioxidant compounds like vitamin E.

Synthesis and SAR of new 5-phenyl-3-ureido-1,5-benzodiazepines as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.

Effects of verapamil on ischaemia-induced impairment of ATP-dependent calcium extrusion in rat heart sarcolemma.

1. The effects of ischaemia and reperfusion were studied on adenosine 5'-triphosphate (ATP)-dependent 45Ca2+-transport in rat heart sarcolemma vesicles. 2. The effect of verapamil, 1 mumol l-1, was studied by pretreatment of the hearts during Langendorff-perfusion and in vitro by adding the drug after isolation of the vesicles. 3. Without drug pretreatment the Ca2+-uptake appeared to be strongly reduced after 30 and after 60 min of global ischaemia, whereas after 30 min of reperfusion it was restored to slightly above the control level. 4. Verapamil pretreatment during the Langendorff perfusion significantly increased Ca2+-uptake in sarcolemma vesicles both before the onset of ischaemia and after 30 min of reperfusion, whereas no beneficial effect was found on the impaired uptake activity during the ischaemic period. 5. When tested in vitro after the isolation of the sarcolemma vesicles, verapamil only inhibited the Ca2+-uptake activity with an IC50 of 112 mumol l-1, which was increased to 250 mumol l-1 after ischaemia and reperfusion. 6. The present study indicates that pretreatment with verapamil, 1 mumol l-1, of the intact rat heart activates an ATP-dependent Ca2+ extrusion process that may contribute to decrease cellular calcium levels in control and, more importantly, in a reperfusion situation. In contrast, in vitro only a less potent inhibition of the extrusion process was found, indicating that physiological regulatory mechanisms may be altered in the vesicles.

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.

Phorbol 12-myristate 13-acetate induces beta-adrenergic receptor uncoupling and non-specific desensitization of adenylate cyclase in human mononuclear leukocytes.

Tumour-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate beta-adrenergic receptor responses in various cell types, presumably by the activation of protein kinase C. In the present investigation we assessed the effect of PMA on the beta-adrenergic receptor-adenylate cyclase system of human mononuclear leukocytes (MNL). It was found that incubation of MNL with PMA resulted in a time- and concentration-dependent desensitization of isoproterenol-induced adenylate cyclase activity. However, the effect of PMA was not restricted to the beta-adrenergic receptor system, since basal adenylate cyclase activity and histamine-, prostaglandin E1-, 5'-guanylylimidodiphosphate (GppNHp)-, and NaF-stimulated values were also reduced. By contrast, no effect was found on the forskolin-induced adenylate cyclase activity. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate had no effect on adenylate cyclase at all, suggesting that the observed PMA effect was specifically mediated by activation of protein kinase C. The reduced beta-adrenergic response induced by PMA was not associated with a reduced beta-adrenergic receptor number, indicating uncoupling of this receptor from adenylate cyclase. Isoproterenol competition curves for 3H-dihydroalprenolol binding to membranes from untreated and PMA-treated cells demonstrated that the uncoupling was due to a reduced ability of the agonist to promote formation of the guanine nucleotide-sensitive high affinity state of the receptor. The results indicate that PMA may cause desensitization of catecholamine-responsive adenylate cyclase in MNL, and that the major locus of alteration is the guanine nucleotide regulatory protein.

Interaction between beta-N-methylamino-L-alanine and excitatory amino acid receptors in brain slices and neuronal cultures.

beta-N-Methylamino-L-alanine (BMAA) stimulated the hydrolysis of polyphosphoinositides (PPI) in hippocampal slices prepared from 8-day old rats. The action of BMAA was antagonized by D,L-2-amino-3-phosphonopropionate (an antagonist of metabotropic receptors) and was largely reduced after lowering the concentration of bicarbonate ions from 25 to 1 mM. In cultured cerebellar neurons, stimulation of PPI hydrolysis by BMAA was mediated by the activation of both metabotropic and N-methyl-D-aspartate (NMDA) receptors. However, BMAA exhibited low activity as an NMDA receptor agonist, as reflected by its low efficacy in increasing cGMP formation in cultures incubated in the absence of extracellular Mg2+. A preferential interaction of BMAA with non-NMDA receptors was confirmed by binding studies on crude synaptic membranes from rat brain. Accordingly, BMAA was more potent in displacing specifically bound [3H]glutamate than 3-(2-carboxypiperazin-4-yl)[1,23H]propyl-1-phosphonic acid (CPP) (a selective NMDA receptor ligand). As expected, the affinity of BMAA for [3H]glutamate or [3H]CPP binding sites was greater in the presence of 25 mM bicarbonate. BMAA weakly displaced specifically bound [3H]glycine in the absence of bicarbonate and, in cultured neurons incubated with buffer containing 1 mM bicarbonate, mimicked glycine in reversing the inhibitory action of kynurenic acid on glutamate-stimulated 45Ca2+ influx. Taken collectively, these results suggest that BMAA acts as a mixed agonist of 'metabotropic' and NMDA receptors.

Modulation of ATP-dependent calcium extrusion and Na+/Ca2+ exchange across rat cardiac sarcolemma by calcium antagonists.

The calcium antagonists verapamil, bepridil, nifedipine and nimodipine inhibited ATP-dependent 45Ca2+ uptake in purified rat ventricular sarcolemma vesicles dose dependently. This inhibition was preceded by a slight stimulation in the case of the two dihydropyridines, but not with bepridil and verapamil. In contrast, Na+/Ca2+ exchange was only inhibited by verapamil and bepridil and not affected by the dihydropyridines. The steepness of the inhibition curves was significantly different for the two processes. No stereoselectivity was found with either process for inhibition by the verapamil enantiomers. Inhibition of the exchange was not due to a decrease of the exchange velocity but to a decrease in exchange capacity. Variation of the antagonist preincubation time did not modify the inhibition of the uptake. The results indicate that two different sites, located at the inner surface of the sarcolemma are involved in the modulation of the ATP-dependent uptake and the Na+/Ca2+ exchange. However, the possibility cannot be ruled out that inhibition of the exchange process is also mediated by an extracellularly located site.

Pharmacological analysis of carboxyphenylglycines at metabotropic glutamate receptors.

Three carboxyphenylglycine derivatives were examined for their activity on glutamate metabotropic receptors negatively linked to adenylate cyclase. Chinese hamster ovary cells stably expressing mGlu2 and mGlu4 were utilised for this study. A receptor binding analysis was also performed for the main classes of glutamate ionotropic receptors and for the glycine binding site on the NMDA-receptor complex. In mGlu2 expressing cells (S)4-carboxy-3-hydroxyphenylglycine and (S)4-carboxy-phenylglycine antagonized forskolin-stimulated cAMP levels, with EC50 of 21 and 970 microM, respectively, acting as agonists at this receptor subtype, whereas (RS) alpha-methyl-4-carboxyphenylglycine antagonized glutamate response in these cells. None of these compounds showed any agonistic or antagonistic activity on mGlu4 expressing cells. No affinity for the ionotropic receptors (NMDA, AMPA and kainate) and for the glycine site of the NMDA-receptor complex was found using the receptor binding approach, except for (RS)4-carboxy-3-hydroxyphenylglycine which showed a pKi of 5.68 in ((+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding for NMDA receptor, although this can be ascribed to the (R) form of the racemic mixture.

Ca2+ channel blocking activity of lacidipine and amlodipine in A7r5 vascular smooth muscle cells.

Inhibition of the K(+)-stimulated increase in cytosolic free Ca2+ by a series of 1,4-dihydropyridines was evaluated in A7r5 vascular smooth muscle cells loaded with the fluorescent Ca2+ indicator fura-2 acetoxymethyl ester. The IC50 of the drugs, added to suspended cells 3 min before 150 mM KCl, gave the following order of potency: lacidipine (2.76 nM) > nitrendipine (3.81 nM) > amlodipine (4.56 nM) > nifedipine (10.08 nM). A7r5 cells were also exposed to the 1,4-dihydropyridines, at their IC50, for 25 min, and then repeated washout cycles were performed before adding KCl. The Ca2+ channel blocking activity of nifedipine and nitrendipine gradually diminished, disappearing after four washout cycles 25, 55, 115 and 175 min after drug treatment. Amlodipine and lacidipine displayed slow onset and offset of antagonism, their activity becoming stronger with time, in spite of the repeated washes. [3H]Lacidipine was avidly and promptly entrapped in A7r5 cells and was not removed by washout. However, its potency as a Ca2+ channel blocker was not directly related to the amount of drug locked in the cell since it increased with time, indicating that lacidipine binds to the lipid bilayer of the cell membrane and then gradually diffuses towards a specific binding site. This model can, therefore, predict the Ca2+ blocking properties of 1,4-dihydropyridines with slow onset and offset of antagonism and could be employed to evaluate compounds selective for vascular smooth muscle.

Stereoisomers of calcium antagonists discriminate between coronary vascular and myocardial sites.

In the retrogradely perfused, paced rat heart, we studied the effects of the stereoisomers of verapamil (VER), gallopamil (GAL), devapamil (DEV) and bepridil (BEP) on the coronary flow and the maximum systolic left ventricular pressure (MSLVP). In addition, the time courses of onset and recovery of these effects were measured. The verapamil analogues showed high stereoselectivity factors (sf) for MSLVP depression in favour of the (-)-enantiomers and low sf's for coronary flow increase. Bepridil showed a low sf for both parameters with the (+)-enantiomer being more potent. In a previous study we found that in the rat heart, dihydropyridine calcium antagonists clearly possess high selectivity for the vascular isochannel site as compared to the myocardial site, whereas racemic verapamil derivatives were devoid of such selectivity. In the present study the (+)-enantiomers of all verapamil congeners revealed a marked vasoselectivity. This was not found for the (-)-isomers, which surprisingly were virtually equipotent for MSLV depression and coronary flow increase, suggesting a different voltage dependence of the two isochannel verapamil sites for the enantiomers of verapamil and its congeners. Onset and offset velocities were clearly different as well. The kinetics of coronary flow increase were identical and fast for all enantiomers studied. MSLV kinetics were slower. In particular the recovery was markedly different for the enantiomers of each drug, the more potent isomer having the lower velocity. Furthermore, the differences in recovery of MSLVP depression between the verapamil type enantiomers suggest that the recovery rate may directly reflect dissociation from the myocardial isochannel verapamil site.

Protection by verapamil and nifedipine against ischaemia-induced loss of [3H]-(+)-PN 200-110 binding sites in the rat heart.

We have studied the effects of 60 min global ischaemia and 30 min of subsequent reperfusion on the binding of [3H]-(+)-PN 200-110 and [3H]-(-)-devapamil (desmethoxyverapamil or D888) in rat heart membranes. The hearts were perfused in the Langendorff-mode and pretreated with 1 mumol/l verapamil, 30 nmol/l and 1 mumol/l nifedipine. After 60 min of global ischaemia in the absence of drugs, we found a reduction of [3H]-(+)-PN 200-110 binding sites, without changes in the equilibrium dissociation binding constant (Kd). After the subsequent reperfusion maximum specific binding (Bmax) was further reduced, whereas the Kd remained constant. [3H]-devapamil binding sites were influenced to a lower extend and showed only a decrease in Bmax at reperfusion. Pretreatment with 1 mumol/l verapamil completely prevented the changes which were observed for [3H]-(+)-PN 200-110. Pretreatment with a low, vasodilating concentration (30 nmol/l) of nifedipine displayed selective protection against the extra reduction in Bmax which was observed during reperfusion. It is concluded that calcium antagonists show protection against the ischaemia-induced loss of dihydropyridine binding sites in relation to their negative inotropic, energy-saving activity. Furthermore, nifedipine at low, vasodilating but not negative inotropic concentrations protects against further reperfusion-induced injury, which protection may be related to an improved flow during reperfusion.

Positive inotropic effects of calcium channel antagonists are not necessarily caused by partial calcium channel agonism.

Recently it has been reported that some dihydropyridine calcium channel antagonists (nifedipine, nimodipine, nitrendipine) are able to produce positive inotropic effects in isolated perfused guinea pig hearts. We studied the effects of nifedipine in isolated perfused paced rat hearts under constant pressure and constant flow perfusion conditions. We found that nifedipine is able to produce a positive inotropic effect under constant pressure conditions but not under constant flow conditions. We conclude that nifedipine does not have partial calcium channel agonistic properties and that the positive inotropic effect seen under constant pressure conditions is a result of the vasodilating properties of the drug. Positive inotropic effects caused by vasodilatation can be explained by the "garden-hose-effect".

Stereoisomers of BAY K 8644 show opposite activities in the normal and ischaemic rat heart. A comparison with nifedipine.

The effects of the stereoisomers and the racemate of the calcium agonist BAY K 8644 and the calcium antagonist nifedipine were studied on the Langendorff-perfused rat heart, subjected to 30 min of global ischaemia. The results show that (-)- and (+/-)-BAY K 8644 induced a strong positive inotropic effect at 100 and 1000 nmol/l and a vasoconstricting effect which was most prominent at 1 and 10 nmol/l, respectively. At higher concentrations the flow reduction was inverted to a flow increase, closely related to the positive inotropic activity. The inotropic status induced by the agonist before the onset of ischaemia was reflected in an accelerated development of the diastolic contracture during ischaemia. During the reperfusion, a complex triphasic effect on the recovery was found, in which probably positive inotropism, vasoconstriction, metabolic and mechanical factors are involved. The (+)-enantiomer of BAY K 8644 behaved as a weak calcium antagonist showing merely vasodilatation, which accelerated the recovery from the ischaemic contracture at reperfusion. The calcium antagonistic, vasodilating effects of the (+)-enantiomer were expressed in the racemate only during the reperfusion phase, where it took an intermediate position between the effects of the (-)- and (+)-enantiomer. In contrast, nifedipine, at negative inotropic - energy saving - concentrations, diminished the height and delayed the development of the energy deprivation-induced left ventricular diastolic contracture during ischaemia. The time needed for recovery from the contracture during reperfusion was significantly shortened already at a 100 times lower, vasodilating concentration of nifedipine.

pH-dependent effects of bepridil on Ca2+-extrusion across rat heart sarcolemma.

The inhibition by bepridil of ATP-dependent Ca2+-uptake and Na+/Ca2+-exchange was studied in isolated rat cardiac sarcolemmal vesicles over a pH range of 6.3 to 7.8. It was found that the protonated and deprotonated species of bepridil induced the same inhibition and no activation of these two transport systems. However, the intrinsic activities of both systems appeared to be strongly dependent on the pH.

Separation and characteristics of inside-out and right side-out vesicles from a rat cardiac sarcolemma preparation.

Purified cardiac sarcolemma (SL) vesicles are highly suitable to study various Ca2+-transport systems present in the SL. We describe in this paper the separation of the Inside-Out (IO) and Right side-Out (RO) oriented vesicle subpopulations from a purified rat heart SL preparation. The isolated subfractions were characterized with respect to the number of beta-adrenergic binding sites and the Ca2+-uptake and (Ca2+-Mg2+)-ATPase activities. It was found that the Ca2+-uptake and the (Ca2+-Mg2+)-ATPase activities reside in the IO fraction and are virtually absent in the RO fraction, confirming that the active Ca2+-uptake represents the outward directed sarcolemmal Ca2+-flux.

Dynamic and kinetic differences of the vascular and myocardial effects of calcium antagonists in the rat heart.

We studied the effects of nifedipine, nimodipine, verapamil, D600 (gallopamil), D888 (desmethoxyverapamil), D890 (quaternized verapamil), bepridil, and diltiazem on the coronary flow and the left ventricular pressure in the retrogradely perfused paced rat heart; in addition, we investigated the time course of onset and recovery of these effects. We found a clear difference in potency order for the vascular and cardiac effects as well as widely different kinetics of coronary flow increase and negative inotropic activity. Furthermore, positive inotropism at low doses of some calcium antagonists seemed to be related to the vascular effects of these compounds. We conclude that the rat heart contains a hydrophylic and readily accessible, vascular "dihydropyridine" site and a more hydrophobic, possibly intramembraneous or intracellular, myocardial "verapamil" site with a lower accessibility for verapamil derivatives and bepridil.

Interaction of lacidipine with its receptor binding site supports a slow onset and long duration of action.

[3H]lacidipine binding to its receptor was characterized to explain its slow onset and long duration of antihypertensive activity. Binding parameters were studied in guinea pig myocardial and cerebral membrane preparations and compared with another dihydropyridine (DHP) calcium antagonist, isradipine. Lacidipine binds competitively to the DHP calcium antagonist receptor of the L-type calcium channel. The binding is allosterically modulated by verapamil and D-cis diltiazem and activated/inhibited by divalent cations. Association and dissociation kinetics of the binding of lacidipine to the receptor were significantly slower than those of isradipine. In addition, the Bmax of lacidipine binding in guinea pig heart microsomes was significantly higher than those of other dihydropyridine calcium antagonist. The results indicate that the slow onset and long duration of action of lacidipine can be explained principally on the basis of the binding characteristics. Although no biphasic receptor binding kinetics could be detected, a fast equilibrium between the receptor and a second compartment, due to the high lipophilicity of lacidipine, cannot be excluded.

Binding of DL-[3H]-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) to rat cortex membranes reveals two sites or affinity states.

A method for measuring [3H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [3H]-AMPA. Non linear curve fitting of [3H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 +/- 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 +/- 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 +/- 0.05 (Kd = 52 nM) and a Bmax of 1.30 +/- 0.23 pmol/mg protein. The pharmacological profile of [3H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [3H]-AMPA binding (IC50 = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272-373 nM). Kainate was a moderate displacer (6.2 microM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 microM, respectively). CPP, GAMS and L-Aspartic acid showed IC50-values of over 400 microM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.