D888 Binding Research Articles

Brain calcium channel related dihydropyridine and phenylalkylamine binding sites in Alzheimer's, Parkinson's and Huntington's diseases

Disruptions of normal calcium (Ca 2+) homeostasis in human aging process, are well documented. The existence of at least four distinct types of Ca 2+ channels, L-, T-, P-, and N-, have been reported in the centra l nervous system. Binding sites for clinically useful dihydropyridines (DPH) and phenylalkylamines (PA) are located on the L-type Ca 2+ channels. In the present study, DHP/[ 3H]PN200-110 and PA/[ 3H](−)-D-888 binding parameters were determined in various brain areas obtained at autopsy from Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) disease patients, and healthy age-matched controls as a means to assess the integrity of L-type channels in these neurological disorders often associated with the aging brain. DHP and PA receptor binding parameters ( K D and B max) were not significantly altered in any of the brain regions studied in AD and PD. However, a significant decrease in the maximal binding capacity of [ 3H]PN200-110 was observed in the striatum of HD patients. Taken together, this suggests that DHP and PA binding sites associated to L-type Ca 2+ channels are mostly preserved in AD and PD brains. Accordingly, the use of DHP- and/or PA-related drugs in these neurological disorders should not be hindered by deficits in their related Ca 2+ channel binding proteins.

Effect of Age and of Hypertrophy on Cardiac Ca2+ Antagonist Binding Sites

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200-110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (-)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax. In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.

Desmethoxyverapamil-sensitive calcium channels in rat portal vein smooth muscle

The whole-cell patch clamp technique was used to analyze the properties of the phenylalkylamine-sensitive calcium channels in smooth muscle cells isolated from the portal vein. (−)-D888 dose dependently inhibited the calcium current elicited from a holding potential of −40 mV (IC 50 = 1.3 nM) in a frequency-dependent manner. No voltage dependence of the inhibition was noted. Independent high- and low-affinity binding sites for (−)-[ 3H]D888 were identified. Calcium entry blockers such as (−)-D888, d-cis-diltiazem and nicardipine completely or partially antagonized the (−)-[ 3H]D888 binding at both types of sites. The properties of this cross-inhibition suggest that phenylalkylamines and d-cis-diltiazem bind at common sites in vascular smooth muscles whereas dihydropyridines bind at distinct sites which are allosterically coupled to the phenylalkylamine sites. As the IC 50 for (−)-D888 found from electrophysiological experiments is not identical to the equilibrium dissociation constants for the high- and low-affinity sites found from binding data (0.47 and 50 nM, respectively), it is suggested that binding of (−)-D888 to both high- and low-affinity sites may be involved in the inhibitory effect of (−)-D888 on calcium channels. Furthermore, these two different binding sites may correspond to two different subtypes of phenylalkylamine-sensitive calcium channels in smooth muscle cells.

Two calcium channels in basolateral membranes of rabbit ileal epithelial cells

The actions of three different types of calcium channel blockers on short-circuit current (Isc) in rabbit ileum were studied. These included the phenylalkylamines, verapamil and (l)-desmethoxyverapamil (D888); the dihydropyridines, nifedipine and nitrendipine; and the benzothiazepine, diltiazem. All of the drugs decreased Isc, a change associated with increased Na and Cl absorption. Verapamil and D888 had the largest effects. The dihydropyridine, BAY K 8644, a calcium channel activator, increased Isc and decreased Na and Cl absorption, effects not inhibited by tetrodotoxin. The phenylalkylamines had an additional effect on Isc in the presence of a maximally inhibitory concentration of the dihydropyridines, suggesting the possibility of two distinct calcium channels, one of which is the L-type voltage-activated, dihydropyridine- and phenylalkylamine-sensitive channel, and the other is a channel only sensitive to phenylalkylamines but not to dihydropyridines. [3H]nitrendipine and [3H]D888 binding to an enriched preparation of basolateral membranes from ileal epithelial cells was characterized. Each ligand bound specifically and saturably to an apparently single population of high-affinity sites with [3H]D888 having three times as many binding sites as [3H]nitrendipine. [3H]nitrendipine binding was partially inhibited by verapamil and D888 and was increased by diltiazem; whereas [3H]D888 binding was inhibited completely by verapamil but only partially by nitrendipine and diltiazem. These transport and binding studies suggest the presence of two types of Ca2+ channels in ileal epithelial cells, one of which interacts with the dihydropyridines, the phenylalkylamines, and the benzothiazepines at three different sites and the other channel that only binds the phenylalkylamines.

The effect of the phenylalkylamine D888 (devapamil) on force and Ca2+ current in isolated frog skeletal muscle fibres.

1. The effects of the (+)- and the (-)-isomer of the phenylalkylamine derivative D888 (desmethoxyverapamil or devapamil) on isometric force and slow Ca2+ inward current were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal glass microelectrodes at 10 degrees C in a TEA sulphate solution containing approximately 4 mM-free Ca2+. 2. In the presence of 0.05-5 microM-(-)-D888 a normal phasic contracture could be induced by a depolarizing voltage step. When depolarization was maintained for some minutes the force-controlling system turned into a stabilized inactivated state (paralysis) from which it recovered upon repolarization within minutes instead of seconds. With the (+)-isomer (0.5-20 microM), a similarly retarded restoration was observed. However, it proved to be less effective than the (-)-isomer. 3. D888 caused a shift to more negative potentials of the S-shaped curve, which describes the voltage dependence of force restoration in the steady state (restoration time 15 min). The potential of half-maximum restoration in the absence of the drug (V = -35.8 mV) changed as follows. (-)-D888: -56 mV (0.05 microM), -69 mV (0.2 microM), -77.5 mV (0.5 microM), and -82 mV (5 microM); (+)-D888: -55.8 mV (0.5 microM), -76.5 mV (5 microM), and -85 mV (20 microM). 4. On the assumption that D888 binds only to the inactivated form of the voltage sensor of force control in the T-tubular membrane (modulated receptor hypothesis) the data presented in paragraph 3 allowed an estimation of the drug-receptor dissociation constants. The KD values ascertained in this way, 1.71 nM for the (-)-isomer and 12.9 nM for the (+)-isomer, are in fair agreement with those obtained from [3H]D888 binding studies by other authors. 5. A comparison between equal concentrations of the two isomers regarding their effect on the speed of restoration and the time needed to transform the sensor into the paralysed state suggests that the differences in the dissociation constants are mainly due to a greater dissociation rate of the (+)-isomer from the sensor. 6. The restoration of the Ca2+ channel was retarded by D888 to a similar extent as that of the voltage sensor. This parallel action on both systems indicates structural similarities between the voltage sensor and the Ca2+ channel. 7. It is concluded that D888 'stabilizes' the inactivated state of the voltage sensor and the Ca2+ channel in a way similar to D600, but with a higher potency. Both isomers of D888 showed an antagonistic action and differed only in their potency.

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.

Native and detergent-solubilized membrane extracts from Drosophila heads contain binding sites for phenylalkylamine calcium channel blockers

Membrane extracts from Drosophila melanogaster heads contain binding sites for [ 3H]D-600, a verapamil-like calcium channel blocker. Scatchard analysis suggested a two binding site model with a dissociation constant ( K d) of 3.3 ± 0.28 nM and a B max of 1.3 ± 0.14 pmol/mg protein for the high affinity site and a K d of 150 ± 28 nM and a B max of 29 ± 10 pmol/mg protein for the low affinity site. The rank order of affinity of phenylalkylamine binding to the high affinity site was (−)-D-888 > (±)-verapamil > (±)-D-600. Displacement of (−)-[ 3H])D-888 binding by phenylalkylamines is stereoselective. The diphenylpiperazine cinnarizine is an effective inhibitor of [ 3H]D-600 binding. However, [ 3H]D-600 binding is only partially inhibited by diltiazem and bepridil at 10 −4M concentrations, and is not affected by dihydropyridine calcium channel drugs at concentrations < 10 −5M. Moreover, only extremely low specific [ 3H]nitrendipine, (+)-[ 3H]PN200-110, or d-(cis)- [ 3H]diltiazem binding was detectable. The phenylalkylamine photoaffinity probe, [N-methyl- 3H]LU 49888, labeled two Proteinase-K sensitive proteins of 136,000 and 27,000 Da. The high molecular weight, trypsin-sensitive band behaves like the high affinity phenylalkylamine receptor, while the low molecular weight, trypsin-insensitive band has properties of the low affinity receptor. Digitonin-solubilized binding activity has a sedimentation coefficient of 12 S on a sucrose gradient.

The Ca2+‐antagonist and binding properties of the phenylalkylamine, anipamil

1. Isolated, Langendorff-perfused rat hearts, isolated membranes, and pharmacological and receptor binding techniques were used to study the properties of the newly developed verapamil derivative, anipamil. 2. When added acutely to isolated, spontaneously beating or electrically paced hearts, anipamil (0.01-0.15 microM) exerted a dose-dependent negative inotropic effect which developed slowly and persisted after 60 min washout. 3. When added acutely (0.05-0.1 microM) to isolated hearts, or when given intravenously (2 mg kg-1 body weight 1 h before the animals were killed), anipamil displaced the dose-response curves for the positive inotropic effect of (0.10-3.0 mM) Ca2+ and (10-50 nM) Bay K 8644 to the right. 4. When added to freshly isolated cardiac membranes, 0.1 microM anipamil increased the dissociation constant (KD) of the phenylalkylamine (-)-[3H]-desmethoxyverapamil ((-)-[3H]-D888) from 1.22 +/- 0.2 to 2.91 +/- 0.46 nM, without any significant change in density (Bmax; control: 163 +/- 17; anipamil: 117 +/- 20 fmol mg-1 protein). Bound (-)-[3H]-D888 was displaceable by (-)-D888 (Ki 1.7 +/- 0.4 nM) greater than (-)-D600 (Ki 12 +/- 0.5 nM) greater than verapamil (Ki 55 +/- 11 nM) greater than (+)-D600 (Ki 108 +/- 12.2) greater than anipamil (Ki 471 +/- 52 nM). 5. In cardiac membranes isolated from rats pretreated with anipamil (2 mg kg-1 i.v.) 1h before they were killed, the KD of (-)-[3H]-D888 binding was increased (P less than 0.05) from 1.59 +/- 0.18 to 3.28 +/-0.65 nM with no significant change in density, compared to the placebo-treated (control) rats. 6. These results establish that anipamil interacts in a competitive manner with the phenylalkylamine binding sites in cardiac membranes, and that it resembles other Ca2+ antagonists in displacing the dose-response curve for the positive inotropic effect of Ca2+ to the right. The results also show that although anipamil binds tightly to the cardiac membranes, it binds to the (-)-[3H]-D888 recognition sites less potently than (-)-D888, (-)-D600 or verapamil.

Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system.

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.

Receptors for diphenylbutylpiperidine neuroleptics in brain, cardiac, and smooth muscle membranes. Relationship with receptors for 1,4-dihydropyridines and phenylalkylamines and with Ca 2+ channel blockade

Neuroleptic molecules of the diphenylbutylpiperidine series (DPBP), such as fluspirilene, penfluridol, pimozide and clopimozide, antagonize binding of (−)[ 3H]desmethoxyverapamil ((−)[ 3H]D888) and (+)[ 3H]PN 200-110 to rabbit brain, heart and smooth muscle membranes. The diphenylbutylpiperidine binding site in all these tissues is distinct but is allosterically related to the 1,4-dihydropyridine binding site and to the binding site. High and low affinity binding sites for (−)D888 were identified. (−)[ 3H]D888 binding at both types of sites was inhibited following the saturation of a single type of diphenylbutylpiperidine binding site. Half-maximal inhibition (K 0.5) of brain, heart and smooth muscle membranes binding by different diphenylbutylpiperidines was in the range of 10–100 nM. These K 0.5 values were one to two orders of magnitude higher than those found for the high affinity diphenylbutylpiperidine receptor in skeletal muscle membranes. The K 0.5 values found in binding experiments in smooth muscle were similar to the (IC 50) values for half-maximal inhibition by diphenylbutylpiperidine of voltage-dependent 45Ca 2+ influx through the slow Ca 2+ channel.

Neuroleptics of the diphenylbutylpiperidine series are potent calcium channel inhibitors.

[3H]Fluspirilene, a neuroleptic molecule of the diphenylbutylpiperidine series, binds to skeletal muscle transverse tubule membranes with a high affinity corresponding to a Kd of 0.11 +/- 0.04 nM, A 1:1 stoichiometry was found between [3H]fluspirilene binding and the binding of (-)-[3H]desmethoxyverapamil [(-)[3H]D888], one of the most potent Ca2+ channel inhibitors. Ca2+ channel inhibitors such as D888, verapamil, gallopamil, bepridil, or diltiazem antagonize [3H]fluspirilene binding besides antagonizing (-)[3H]-D888 binding. Neuroleptics, especially those of the diphenylbutylpiperidine family, also antagonize both (-)[3H]D888 binding and [3H]fluspirilene binding. There is an excellent correlation between affinities found from [3H]fluspirilene binding experiments and those found from (-)[3H]D888 binding experiments. Analysis of the properties of these cross-inhibitions indicates that [3H]fluspirilene binds to a site that is not identical to that for phenylalkylamine derivatives (gallopamil, verapamil, diltiazem, and bepridil). Voltage-clamp experiments have shown that fluspirilene is an efficient inhibitor of the voltage dependent Ca2+ channel, achieving a half-maximal effect near 0.1-0.2 nM and nearly complete blockade at 1 nM. Fluspirilene blockade has little voltage dependence.

Characterization of binding sites for two classes of calcium channel antagonists in human forebrain

1,4-Dihydropyridine ([ 3H]PN200-110) and phenylalkylamine ([ 3H](−)D-888) bound with high affinity to human brain cortex homogenates. The ligand selectivity pattern indicates that [ 3H]PN200-110 binding was inhibited by related 1,4-dihydropyridine analogues but not phenylalkylamines. On the other hand, phenylalkylamines were potent inhibitors of [ 3H](−)D-888 binding in human brain. The radioautographic distribution of [ 3H]PN200-110 and [ 3H](−)D-888 binding sites demonstrates that 1,4-dihydropyridine and phenylalkylamine sites were similarly distributed in human forebrain.

Calcium Entry Blockers: Autoradiographic Mapping of Their Binding Sites in Rat Brain

This chapter discusses a possible functional role of organic calcium entry blockers in the brain, and performs receptor autoradiographic studies in this tissue, using ( +)-[ 3 H]PN200-110, ( ±)-[ 3 H]D600 and (-)-[ 3 H]D888 as ligands. The results of such studies can point to the brain areas that contain a high density of these sites that could then be used in the studies aimed at detecting a possible functional role of voltage-dependent calcium channels (VDCC) in the brain. It is observed in (+)-I3H] PN200-110 binding that dopaminergic or noradrenergic neurons, at least in the areas of their cell bodies, are perhaps not the prime candidates for the studies of a functional role of VDCC in the brain. The pharmacological properties as well as the pattern of the distribution of ( ±)-[3H]D600 binding sites, as revealed by receptor autoradiography, are difficult to reconcile with the proposal that ( ±)-[3H]D600, can be used in all the brain areas as a specific probe for the studies of the phenylalkylamine drug receptors of VDCC. It is observed that (-)-[ 3 H]D888 binding sites can be detected in all those brain areas that contain high affinity binding sites for (+)-[ 3 H]PN200-110.