Interaction Of Diltiazem Research Articles

Interaction of diltiazem with an intracellularly accessible binding site on CaV1.2

BACKGROUND AND PURPOSEDiltiazem inhibits CaV1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits.EXPERIMENTAL APPROACHCaV1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through CaV1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).KEY RESULTSQuaternary derivative d-cis-diltiazem inhibited CaV1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of CaV1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.CONCLUSION AND IMPLICATIONSThe data show that use-dependent block of in CaV1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.

Potentiating effect of diltiazem on pentobarbital-induced hypnosis is augmented by serotonergic system: The TMN and VLPO as key elements in the pathway

To investigate the mechanism by which L-type Ca 2+ channel blockers exerted potentiating effects on pentobarbital-induced hypnosis, the present study was undertaken to determine if the interaction of diltiazem and serotonergic system influences the architecture of pentobarbital sleep in rats and examined c-Fos expression in the ventrolateral preoptic nucleus (VLPO) and the tuberomammillary nucleus (TMN). The polysomnogram consisting of EEG and EMG was recorded for analyzing sleep architecture. The results showed that diltiazem (2.0 and 5.0 mg/kg, p.o.) increased both total pentobarbital sleep and slow wave sleep (SWS), but decreased rapid eye movement (REM) sleep. These effects were potentiated by 5-hydroxytryptophan (5-HTP), a precursor of serotonin, but abolished by p-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase. Diltiazem (1 mg/kg, p.o.) or 5-HTP (2 mg/kg, i.p.) alone did not change the architecture of pentobarbital sleep and pentobarbital-induced c-Fos expression in the VLPO and the TMN, but co-administration of them significantly increased both total pentobarbital sleep and SWS, whereas decreased REM sleep, with increasing c-Fos expression in the VLPO and concomitantly decreasing c-Fos expression in the TMN. These findings indicate that the serotonergic system may be involved in the augmentative effect of diltiazem on pentobarbital sleep and the VLPO–TMN neuronal circuit may play a key role.

Diltiazem causes open channel block of recombinant 5-HT3 receptors.

1. To extend our knowledge of the site and mechanism of action of L-type Ca2+ channel antagonists on 5-HT3 receptors, whole-cell voltage clamp electrophysiology was used to investigate the action of one of these compounds, diltiazem, on the recombinant receptor expressed in human embryonic kidney (HEK) 293 cells. 2. Application of diltiazem with 5-HT (30 microM) caused an increase in the rate of receptor current decay, but did not significantly affect peak current (Ip), the EC50 or the Hill coefficient, indicating a non-competitive mechanism of action. Pre-application of the antagonist had no effect indicating that diltiazem mediates its effects by binding preferentially to the open state of the 5-HT3 receptor. 3. To examine the effects of diltiazem on the open state of the receptor in more detail we used 10 mM 5-hydroxyindole (5-OHi) to reduce receptor desensitisation. These experiments showed that diltiazem causes a rapid, reversible, block in the presence of agonist but can become trapped in the unliganded state of the receptor by prior washout of agonist. Dose-inhibition data yielded an IC50 of 5.5 microM and a Hill coefficient of 0.96; inhibition was slightly voltage dependent as the degree of blockade at +60 mV was reduced. 4. The Hill coefficient of near unity suggests a single molecule of diltiazem mediates inhibition and, indeed, kinetic analysis verified that the interaction of diltiazem with the 5-HT3 receptor was well described by a bimolecular reaction scheme. The results suggest that diltiazem acts by causing open-channel block of the 5-HT3 receptor.

Interaction of calcium channel antagonists with calcium: spectroscopic and modeling studies on diltiazem and its Ca2+ complex.

Using spectral techniques, the solution conformation of diltiazem was studied in acetonitrile with special reference to the effect of Ca2+ on the drug structure. Complete assignment of the proton resonances in the 1H-NMR spectrum of the drug was made using one-and two-dimensional spectral analyses. A two-dimensional 1H-NOESY spectrum (in the phase-sensitive mode) was obtained to identify the interproton connectivities in the drug molecule. A molecular modeling program involving Monte Carlo simulation and energy minimization was employed to arrive at the structure of the drug. The program was run with and without the input of the interproton distances derived from the NOESY cross peaks. Both the protocols led to a structure of the drug which was generally similar to that reported from X-ray diffraction data on crystalline diltiazem hydrochloride (Kojic-Prodic, et al. Helv. Chim. Acta 1984, 67, 916-926). However, significant differences between the two structures were seen in the orientations of the substituent groups attached to the benzothiazepine ring. Substantial changes in the circular dichroic (CD) and 1H-NMR spectra of diltiazem were observed on addition of Ca2+ up to a mole ratio of 0.5 Ca2+ per drug. Relatively large changes were seen in 1H resonances of the N-methyl protons and the methylene protons attached to the heterocyclic nitrogen. Analysis of the binding isotherms from CD data at 22 +/- 1 degrees C indicated a 2:1 drug:Ca2+ "sandwich" complex with an estimated dissociation constant of 140 microM. One-dimensional difference NOE and two-dimensional NOESY spectra revealed interproton connectivities between two drug molecules that were compatible with the sandwich complex formation. The interproton distances derived from the volume integrals of the NOESY cross peaks were used as geometrical constraints in modeling the Ca(2+)-bound conformation of diltiazem. The minimum-energy conformation corresponded to the sandwich complex where Ca2+ was coordinated to three oxygens in each of the two drug molecules. Combined with our earlier data on the ability of diltiazem to translocate Ca2+ across the lipid bilayer in synthetic liposomes (Ananthanarayanan, V.S.; Taylor, L.; Pirritano, S.Biochem. Cell Biol. 1992, 70, 608-612), the structural data presented here point to a role for Ca2+ in the interaction of diltiazem with its membrane-bound receptor.

3H]-nitrendipine binding in normal and cardiomyopathic hamster hearts: Modulation by temperature, verapamil and diltiazem

The characteristics of high affinity dihydropyridine binding sites were compared in normal and cardiomyopathic hamster hearts to probe for possible defects in the calcium channel which could lead to calcium overload and, in turn, to the muscle necrosis characteristic of cardiomyopathy. Kinetic studies of the temperature dependence of [3H]-nitrendipine binding to ventricular homogenates from 60-day-old normal and cardiomyopathic hamsters showed that, in normal hamsters, the rate of dissociation (0.049 +/- 0.006/min at 25 degrees C) was highly temperature-dependent (Q10 = 4.40 +/- 0.69) and that neither the rate nor the temperature dependence was influenced by disease. The rate of association (1.12 +/- 0.11/min/nM at 25 degrees C) was weakly temperature-dependent (Q10 = 1.25 +/- 0.04) and similarly unaffected by disease. The rate of dissociation of [3H]-nitrendipine was increased by verapamil and decreased by diltiazem with little effect on the association rate. Allosteric interactions of diltiazem and verapamil with the dihydropyridine receptor were identical in normal and cardiomyopathic hearts and, together with the normal temperature sensitivity, show that there is no abnormality at the related binding sites for nitrendipine, verapamil and diltiazem in the calcium channel of the cardiomyopathic heart.

Interaction of diltiazem with propranolol on atrioventricular conduction and refractoriness in the dog.

Propranolol pretreatment (0.1 mg X kg-1) significantly increased the lengthening induced by diltiazem (0.15 mg X kg-1 + 0.01 mg X kg-1 X min-1, 20 min) of the A-H interval of His bundle potential recordings in the dog. In the presence of propranolol, diltiazem also significantly increased the atrioventricular effective refractory period. The results suggest the possible occurrence of AV blocks as a result of the use of a diltiazem-beta blocker combination in clinical practice.