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.

Cycloalkyl indole-2-carboxylates as useful tools for mapping the "north-eastern" region of the glycine binding site associated with the NMDA receptor.

A novel series of indole-2-carboxylate analogues of GV 150526 (1) in which the terminal phenyl ring belonging to the side chain present in the position C-3 has been replaced with a bridged cycloalkyl group was synthesized and evaluated for its pharmacological profile. Modelling studies on this class of novel glycine antagonist allowed us to identify an asymmetric lipophilic pocket present in the "North-Eastern" region of the pharmacophoric model of the glycine binding site associated to the NMDA receptor. Among the derivatives prepared, 3-[2-(1-adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2 -carboxylic acid 6b and 3-[2-(norbornylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-c arboxylic acid 6l were found to be antagonists acting at the strychnine-insensitive glycine binding site, showing nanomolar affinity for the glycine binding site (Ki = 63 and 19 nM, respectively), coupled with high glutamate receptor selectivity (IC50 > 10(-5) M at the NMDA, AMPA, KA binding sites) and high in vivo potency after systemic administration by inhibition of convulsion induced by NMDA in mice.

[3H]5,7-dichlorokynurenic acid recognizes two binding sites in rat cerebral cortex membranes.

Binding of [3H]5,7-dichlorokynurenic acid ([3H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [3H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pKD value of 7.24 (KD = 57.5 nmol/l) and a maximum binding value (Bmax) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [3H]DCKA and [3H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [3H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carb oxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pKi = 8.24, Ki = 5.6 nmol/l). The low affinity component of [3H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [3H]DCKA binding site remains to be elucidated.

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.

Synthesis and receptor binding affinity of cholecystokinin receptor ligands: 2-and 1-indolyl derivatives of PD134308.

The synthesis of two "dipeptoids" structurally related to the CCK-B antagonist CI-988 (PD134308) is described. The 2- and 1-indolyl derivatives 4a, b were prepared in order to define the role of the tryptophan moiety in this series of "dipeptoids". They were evaluated as competitors in the binding of [3H]-CCK8S on guinea pig brain CCK-B receptors.

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.

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.

A further analysis of the contraction induced by activation of cholecystokinin A receptors in guinea pig isolated ileum longitudinal muscle-myenteric plexus.

The activity of a selective cholecystokinin (CCK)-A receptor agonist, N-acetyl derivative of A71623 (Ac-Trp-Lys(epsilon-N-[2-methylphenylamino-carbonyl]) -Asp-(NMe)Phe-NH2) was investigated in the guinea pig isolated ileum longitudinal muscle myenteric plexus. NAA caused both a phasic and tonic contraction at all concentrations tested (1-1000 nM). The selective CCK-A antagonist L-364,718 (Devazepide) antagonized both types of contraction with a pKB of 10.10 and 9.95, respectively. The CCK-B selective antagonist L-365,260 ((3R(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1, 4-benzodiazepine-3yl)-N-(3-methylphenyl)-urea) was inactive up to a concentration of 30 nM. Atropine at 300 nM and 1000 nM reduced the maximal response of NAA by only 17% and 50%, respectively. The selective neurokinin (NK)-1 antagonists GR 82334 ([D-pro9[Spiro-gamma-Lactam] Leu10, Trp11]-Phys (1-11)9) at 300 and 1000 nM and (+-) CP-96,345 [(2S, 3S)-cis- 2-(diphenylmethyl)-N- [(2-methoxyphenyl)-methyl] -1-azabici-clo [2.2.2]octan-3-amine] at 10 nM were inactive or partially active. When atropine and GR 82334 or (+/-) CP-96,345 were combined, they produced a dose-dependent synergistic inhibition of both phasic and tonic contractions induced by NAA. The selective NK-3 receptor agonist senktide induced both phasic and tonic contractions that were blocked by tetrodotoxin. In the presence of atropine and GR 82334, both 300 nM, a synergistic depression of the response to senktide similar to that observed for the agonist NAA was disclosed.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric modulation of [3H]CGP 39653 binding by glycine in rat brain.

D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a KD of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [3H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [3H]CPP binding but inhibits [3H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [3H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [3H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [3H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [3H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.

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.

3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid recognizes two N-methyl-D-aspartate binding sites in rat cerebral cortex membranes.

Binding of 3-[(+-)-2-carboxypiperazin-4-yl][3H]-propyl-1-phosphonic acid ([3H]CPP), a competitive inhibitor of N-methyl-D-aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high-affinity binding site with a pKD value of 7.53 +/- 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 +/- 0.36 pmol/mg of protein, and a low-affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low-affinity binding site may represent an agonist-dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high-affinity binding site may be linked to an antagonist-preferred receptor, for which no function has yet been reported.

Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells.

One of the major biological targets of free radical oxidations, prone, for anatomical reasons, to oxidative challenges, is the cardiovascular system. In the present paper the effect of hydrogen peroxide on intracellular ionized calcium ([Ca2+]i) homeostasis in smooth muscle cells (SMC) is studied, the major aim of the study being a better understanding of the protective effect of antioxidants and Ca2+ channel blockers. The exposure of SMC to 300 microM H2O2 induced a rapid increase of [Ca2+]i, followed by a decrease to a new constant level, higher than the basal before the oxidative challenge. When incubation medium was Ca2+ free, the pattern of [Ca2+]i change was different. The rapid increase was still observed, but it was followed by a rapid decrease to a level only slightly above the basal before the oxidative challenge. The involvement of intracellular Ca2+ stores was tested by using vasopressin, a hormone able to induce discharge of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. When H2O2 was added after vasopressin no [Ca2+]i increase was observed. Treatment of cells, in which the stable increase of [Ca2+]i was induced by H2O2, with disulfide reducing compounds, induced a progressive decrease of [Ca2+]i toward the level observed before the oxidative challenge. Calcium channel blockers and antioxidants, on the other hand, effectively prevented the stabilization of [Ca2+]i at the high steady-state, after the internal Ca2+ release phase. Dihydropyridine Ca2+ channel blockers were by far more active than verapamil and among those the most active was lacidipine. Also the antioxidants trolox and N,N'-diphenyl-1,4-phenylenediamine both prevented the [Ca2+]i unbalance. These results suggest that Ca+ channel blockers and antioxidants, although inactive on oxidative stress-induced Ca2+ release from intracellular stores, prevent the increased influx apparently related to a membrane thiol oxidation.

Evidence for two cholecystokinin receptors mediating the contraction of the guinea pig isolated ileum longitudinal muscle myenteric plexus.

In guinea pig isolated ileum longitudinal muscle myenteric plexus, cholecystokinin octapeptide (CCK-8S) produced a rapid (phasic) contraction followed by a slower tonic phase. The tetrapeptide derivative CCK-4 and pentagastrin elicited only the phasic response up to 10(-6) M, whereas the tonic phase was also apparent at higher concentrations. The rank order of potency for the effect of agonists on the tonic and phasic responses were CCK-8S much greater than gastrin greater than CCK-8US congruent to pentagastrin greater than CCK-4 and CCK-8S greater than gastrin congruent to pentagastrin greater than CCK-4 greater than CCK-8US, respectively. Phasic responses of CCK-8S and CCK-4 were sensitive to atropine, whereas the tonic response could be completely abolished with the neurokinin-1 antagonist GR82334. The CCK-A receptor antagonist L-364,718 up to 10(-7) M had little effect on the phasic contracture of CCK-4. The CCK-B/gastrin receptor antagonist L-365,260 had no effect on the CCK-8S phasic response up to 10(-7) M, but antagonized the phasic response induced by low concentrations of CCK-4 in a competitive manner with an estimated pKB of 8.51. This value is close to that of 8.53 found in a guinea pig cortical binding assay. Both the second phase of the CCK-4 phasic concentration response curve (CRC) and the tonic contraction were insensitive to L-365,260.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Calcium antagonists show two modes of protection in ischemic heart failure.

Retrogradely perfused, isovolumically beating, paced rat hearts were subjected to 30 min of global ischemia in the absence and presence of nifedipine, verapamil, bepridil and quaternary bepridil at a concentration ranging from 3 to 10,000 nmol/l. Under constant pressure conditions, the arrest of coronary flow and the reduction of ventricular contraction during global ischemia were readily reversible and quickly returned at reperfusion. During ischemia, however, a diastolic contracture developed, which was slowly reversible upon reperfusion. The calcium antagonists studied appeared to delay and diminish in a concentration-dependent way the diastolic contracture during ischemia. Furthermore, they accelerated the reduction of this contracture at reperfusion. Nifedipine, bepridil and quaternary bepridil showed a 100 to 1000 times higher potency in accelerating the recovery of the diastolic contracture during the reperfusion phase than in reducing the development of diastolic tension during the ischemic period itself, whereas verapamil hardly discriminated these two phases. When the hearts were reperfused with nifedipine under constant flow conditions, reduction of the diastolic contracture during ischemia could still be observed, but the accelerated reduction of end-diastolic pressure during reperfusion was no longer present. The results are discussed in relation to the energy saving, negative inotropic activity of the drugs before the ischemic period and the strong coronary vasodilation, which seems to be involved in the protective activity of the drugs, especially during reperfusion.

Stereoisomers of calcium antagonists distinguish a myocardial and vascular mode of protection against cardiac ischemic injury.

Concentration-dependent effects of the enantiomers of the calcium antagonists, gallopamil, diltiazem, and bepridil have been studied in the Langendorff-perfused rat heart, subjected to 30 min of global ischemia. It is shown that the time course, as well as the height of the energy deprivation-induced left ventricular diastolic contracture that develops during ischemia, can be selectively inhibited by negative inotropic concentrations of the calcium antagonist enantiomers. The time needed for recovery from the diastolic contracture during the reperfusion phase can be shortened significantly by lower, vasodilating concentrations of the drugs. In normoxically perfused hearts, stereoselectivity factors (sf) of the enantiomers of the compounds amounted to 63, 10, and 2 for the negative inotropic and 12.6, 79, and 4 for the vasodilating activities of gallopamil, cis-diltiazem, and bepridil, respectively. The sf values of negative inotropism proved to be remarkably similar to sf values of 50 and 7.9 for gallopamil and cis-diltiazem in the protection of the ischemic contracture during ischemia, whereas the sf values of coronary flow increase closely paralleled the values of 7.9, 63, and 2.5 for gallopamil, cis-diltiazem, and bepridil, respectively, in protection during the reperfusion phase. The results strongly suggest that at reperfusion the vasoselective enantiomers of calcium antagonists provide protection related to improved tissue perfusion, and thereby possibly restoring the distorted ionic and energetic homeostasis, whereas the other enantiomers are more involved in a direct energy-saving activity, resulting in protection during the ischemic period.