D-cis Diltiazem Research Articles

Genetic dissection of horizontal cell inhibitory signaling in mice in complete darkness in vivo.

To test the hypothesis that horizontal cell (HC) inhibitory signaling controls the degree to which rod cell membranes are depolarized as measured by the extent to which L-type calcium channels (LTCCs) are open in complete darkness in the mouse retina in vivo. Dark-adapted wild-type (wt), CACNA1F (Ca(v)1.4(-/-)), arrestin-1 (Arr1(-/-)), and CACNA1D (Ca(v)1.3(-/-)) C57Bl/6 mice were studied. Manganese-enhanced MRI (MEMRI) evaluated the extent that rod LTCCs are open as an index of loss of HC inhibitory signaling. Subgroups were pretreated with D-cis-diltiazem (DIL) at a dose that specifically antagonizes Ca(v)1.2 channels in vivo. Knockout mice predicted to have impaired HC inhibitory signaling (Ca(v)1.4(-/-) or Arr1(-/-)) exhibited greater than normal rod manganese uptake; inner retinal uptake was also supernormal. Genetically knocking out a closely associated gene not expected to impact HC inhibitory signaling (CACNA1D) did not generate this phenotype. The Arr1(-/-) mice exhibited the largest rod uptake of manganese. Manganese-enhanced MRI of DIL-treated Arr1(-/-) mice suggested a greater number of operant LTCC subtypes (i.e., Ca(v)1.2, 1.3, and 1.4) in rods and inner retina than that in DIL-treated Ca(v)1.4(-/-) mice (i.e., Ca(v)1.3). The Ca(v)1.3(-/-) + DIL-treated mice exhibited evidence for a compensatory contribution from Ca(v)1.2 LTCCs. The data suggest that loss of HC inhibitory signaling is the proximate cause leading to maximally open LTCCs in rods, and possibly inner retinal cells, in mice in total darkness in vivo, regardless of compensatory changes in LTCC subtype manifested in the mutant mice.

The Hepatoprotective Effect of Diltiazem and Silymarin

The clinical use of diltiazem has been suggested to be a reasonable approach for combating free radical induced liver damage. The hepatoprotective effect was observed with d-cis diltiazem but not with l-cis diltiazem, suggesting that diltiazem is stereospecific in protecting against lipid peroxidation using isolated microsomes. In the present work we examined d-cis diltiazem’s antioxidant property using Chang and PLC hepatocyte cultures, and the combination of diltiazem and silymarin since silymarin itself has been reported to possess hepatoprotectant properties. Diltiazem (5 and 10 μM) significantly reduced free radical levels, as did silymarin. The combination of diltiazem and silymarin further protected cells against oxidative stress (p<0.001) compared to either drug alone. Diltiazem, silymarin and the combination were associated with enhanced ATP and reduced Bax and Bax mRNA levels in both cell types with the combination drug treatment showing a much greater difference. The combination of dilitiazem and silymarin further enhanced cell viability. We conclude that low dose diltiazem and silymarin can function as a hepatoprotectant against free radical damage due to oxidative stress. The protective nature extends to reducing levels of the pro-apoptotic Bax protein.

Pharmacological strategies to block rod photoreceptor apoptosis caused by calcium overload: a mechanistic target-site approach to neuroprotection.

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited, and disease-, injury- and chemical-induced retinal degeneration. Our aims were three-fold: 1) to determine the kinetics of rod apoptosis and Ca2+ overload in Pde6b9rd1) mice and developmentally lead-exposed rats, 2) to establish a pathophysiologically-relevant model of Ca2+ overload/rod-selective apoptosis in isolated rat retina and 3) to examine different mechanistic based neuroprotective strategies that would abrogate or mollify rod Ca2+ overload/apoptosis. Retinal morphometry and elemental calcium content ([Ca]) determined the kinetics of rod apoptosis and Ca2+ overload. A multiparametric analysis of apoptosis including rod [Ca], a live/dead assay, rod oxygen consumption, cytochrome c immunoblots and caspase assays was combined with pharmacological studies of an isolated rat retinal model of rod-selective Ca2+ overload/apoptosis. Ca2+ overload preceded rod apoptosis in mice and rats, although the extent and kinetics in each differed significantly. The isolated rat model of rod Ca2+ overload/apoptosis showed that blockade of Ca2+ entry through rod cGMP-activated channels with L-cis diltiazem was partially neuroprotective, whereas blockade of Ca2+ entry into rods through L-type Ca2+ channels with D-cis diltiazem or verapamil provided no protection. Inhibition of the mitochondrial Na+/Ca2+ exchanger with D-cis diltiazem provided no protection. CsA and NIM811, mitochondrial permeability transition pore (mPTP) inhibitors, blocked all Ca(2+)-induced apoptosis, whereas the caspase-3 inhibitor DEVD-fmk only blocked the downstream cytochrome c-induced apoptosis. The successful pharmacological neuroprotective strategies for rod Ca2+ overload/apoptosis targeted the rod cGMP-activated channels or mPTP, but not the rod L-type Ca2+ channels.

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.

Differential potentiation by calcium antagonists of neuromuscular blockade induced by pancuronium and succinylcholine in cats in vivo.

The effects of several calcium antagonists (verapamil, nicardipine and two diltiazem isomers, d-cis and l-cis diltiazem) alone and associated to non-depolarizing (pancuronium) and depolarizing (succinylcholine) neuromuscular blockers, were evaluated on sciatic nerve-tibialis anterior muscle preparations from cats in vivo. The calcium antagonists used (at 0.1 and 0.5 mg/kg iv) did not modify the height of muscular twitches elicited indirectly. However, these agents potentiated in a dose-dependent way the neuromuscular blockade induced by iv pancuronium (2-40 micrograms/kg) and succinylcholine (6-200 micrograms/kg). The order of potency in increasing the effects of pancuronium was nicardipine much greater than d-cis diltiazem greater than or equal to verapamil, whereas the order of potency in enhancing succinylcholine effects was d-cis diltiazem greater than or equal to verapamil much greater than nicardipine. The effects of diltiazem were stereoselective, thus the potentiation induced by d-cis diltiazem was significantly greater in all cases than that induced by l-cis diltiazem, which suggests that calcium channel blockade plays a role in these interactions. However, other mechanisms such as calcium antagonists-induced nicotinic receptor desensitization may also be involved.

Dihydropyridine Binding Sites in Aerobically Perfused, Ischemic, and Reperfused Rat Hearts

The time course of normothermic (37 degrees C) and hypothermic (22 degrees C) global ischemia, and of post-ischemic reperfusion on the affinity (KD), selectivity, and density (Bmax) of the high-affinity 1,4 dihydropyridine (DHP) calcium channel antagonist binding sites were studied in isolated rat heart membranes, using (+)[3H]PN 200-110. Short periods (10 or 20 min) of ischemia at either 37 degrees C or 22 degrees C altered neither the KD nor Bmax of these binding sites as compared with aerobic controls. By contrast, longer periods of normothermic ischemia (30 or 60 min) caused a reduction (p less than 0.05) in Bmax (36% after 30 min and 32.7% after 60 min) without change of KD. This reduction in Bmax was reversed by 15-min reperfusion at 37 degrees C after 30 but not 60-min ischemia. There was no effect of ischemia (up to 60 min) on the density of DHP binding sites under hypothermic conditions. Irrespective of the experimental conditions the selectivity of the binding sites was maintained, with (+)PN 200-100 greater than (-)Bay K8644 greater than (-)PN 200-110 = (+)Bay K8644 much greater than (-)D600 in displacing (+)[3H]PN 200-110, and D-cis diltiazem stimulating the binding. These results show that the ischemia-induced change in Bmax is specific, reversible, and time- and temperature-dependent.

Voltage-dependent charge movement in skeletal muscle is selectively increased by L-and D-cis diltiazem

Voltage-dependent charge movement in skeletal muscle is selectively increased by L-and D-cis diltiazem

Cyclandelate as a calcium modulating agent in rat cerebral cortex.

Cyclandelate is clinically effective in a variety of cerebrovascular indications, but its precise mode of action is unclear. Hence, this study investigated the interaction of cyclandelate, cyclandelate alcohol and cyclandelate acid with the binding sites for radioactively labelled 3H-nitrendipine, a Ca++ entry blocker of the 1,4-dihydropyridine type, on rat cerebral cortex membranes. Cyclandelate showed a dissociation constant (Kd) of 7.1 +/- 1.4 X 10(-5) mol/L (35% inhibition of 3H-nitrendipine binding at 2 X 10(-4) mol/L cyclandelate), cyclandelate alcohol had a Kd value of 1.7 +/- 0.1 X 10(-4) mol/L (maximal 70% inhibition of 3H-nitrendipine binding) whereas cyclandelate acid was inactive. For comparison, nifedipine (Kd of 2.6 +/- 0.3 X 10(-9) mol/L inhibition of 68% of 3H-nitrendipine binding), d-cis diltiazem (Kd of 1.1 +/- 0.1 X 10(-7) mol/L enhancement of 39% of 3H nitrendipine binding) and +/- -verapamil [Kd values of 1.4 +/- 0.4 X 10(-7) mol/L (38% inhibition) and 5.3 +/- 1.7 X 10(-4) mol/L (62% inhibition)] were used. Thus, cyclandelate may exert its clinical activity in cerebral ischaemia or hypoxia at least in part through a calcium modulatory effect.

Calcium Channels

The structural features and molecular properties of calcium channels have been explored with 1,4-dihydropyridines, d-cis diltiazem and verapamil in radiolabeled form. The concept of multiple (at least three) drug receptor sites of the channel (sites 1, 2, and 3) has been proposed. Site 1 is labeled by 1,4 dihydropyridines, site 2 by (+/-) verapamil (in skeletal muscle) and by (-) desmethoxyverapamil (in brain, skeletal muscle, and heart), site 3 by d-cis diltiazem (in skeletal muscle). The three distinct drug receptor sites communicate with each other via reciprocal allosterism and are linked to divalent cation binding sites. The concept of the reciprocal allosteric communication between the receptor sites has been proven by individual labeling of each of the drug receptor sites in the skeletal muscle t-tubule membrane where the density of calcium channels is extremely high. In brain and heart, labeling of the receptor sites 1 and 2 has also been successful. Three novel ligands for the calcium channel are presented: 125I-iodipine, a 1,4 dihydropyridine ligand, which has an exceptionally high specific activity (2,200 Ci/mmol), 3H-azidopine, a high-affinity, photoaffinity ligand and finally (-) 3H-desmethoxyverapamil, a high-affinity radioligand for receptor site 2. These ligands will aid in the isolation and final characterisation of the calcium channel. 125I-iodipine, 3H-azidopine and (-) 3H-desmethoxyverapamil bind to detergent-solubilized channels from skeletal muscle t-tubules. Whereas the 1,4-dihydropyridines label only a 12.9 S macromolecule, (-) 3H-desmethoxyverapamil additionally labels a approximately 5.0 S peak, where almost no high-affinity 1,4 dihydropyridine binding is found. Taken together with the results from target-size analysis experiments and photoaffinity labeling, an oligomeric structure of the channel is most likely. One polypeptide chain (Mr = 145,000) in reduced form has been unambiguously identified and is currently further characterized. It remains to be seen if the all of the earlier postulated four elements of the channel (alpha, beta, delta, gamma) are on different polypeptide chains or if the 145,000 peptide corresponds to the (beta, delta) or the (delta, gamma) element.

Solubilization and affinity labeling of a dihydropyridine binding site from skeletal muscle: Effects of temperature and diltiazem on [ 3H]dihydropyridine binding to transverse tubules

Effects of temperature and d-cis-diltiazem (DTZ) on [ 3H]nitrendipine (NTD) and [ 3H]nimodipine (NIM) binding to skeletal muscle t-tubular membranes were studied. A decrease in temperature from 37°C to 10°C decreased K D and increased B max slightly. DTZ increased binding by increasing B max under all conditions and also decreased K D for NTD at 37°C. The binding protein labeled with [ 3H]isothiocyanate dihydropyridine revealed a molecular weight of 36,000. The binding site for NTD was solubilized by deoxycholate and dihydropyridine binding was still stimulated by DTZ in the solubilized form.

High affinity specific [ 3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [ 3H]( +)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [ 3H]( +)PN 200-110 was saturable, reversible, and of high affinity (K d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [ 3H]( +)PN 200-110, the dissociation constant of [ 3H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [ 3H]( +)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [ 3H]( +)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [ 3H]( +)PN 200-110 binding were due mainly to alterations in the dissociation constant (K d), without alterations in the binding density (B max). The new [ 3H]( +)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [ 3H]nitrendipine at physiological temperatures. [ 3H( +)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.