Biomedical subjects

S L Hamilton

Publications and source records attributed to S L Hamilton.

At least 55 records · Page 3Linked to original sources

PubMed1990

Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel.

In this paper, we study the modulation of the rabbit fast twitch skeletal muscle calcium release channel by assaying the kinetics of [3H]ryanodine binding, 45Ca2+ flux, and single-channel activity. The effects of modulators of the Ca2+ release channel (confirmed here with both flux and single-channel data) were examined for effects on [3H]ryanodine binding to terminal cisternae vesicles. We find that activators of the release channel, such as adenine nucleotides (1 mM) and caffeine (1 mM), enhance the rate of association of [3H]ryanodine, whereas inhibitors, such as Mg2+ (1 mM) and ruthenium red (100 nM), decrease the rate of association. High concentrations of either ryanodine or ruthenium red, which close the channel, slow the dissociation of [3H]ryanodine, suggesting that at these concentrations the inhibitory effects of both ryanodine and ruthenium red occur as the result of binding at a site distinct from but interacting cooperatively with the high affinity site. Our data are consistent with a model in which the high affinity ryanodine binding site is within a conformationally sensitive area of the channel, such that conditions that open the channel (ATP, caffeine, etc.) enhance the rate at which [3H]ryanodine reaches its binding site and other conditions that close the channel (the binding of ryanodine and ruthenium red to a low affinity site) slow the dissociation of [3H]ryanodine from the high affinity site. Some conditions that inhibit channel activity (high concentrations of Mg2+ and Ca2+) slow association but do not affect dissociation of bound [3H]ryanodine, suggesting a completely different state of the channel from that which is inactive in the presence of high concentrations of ryanodine or ruthenium red. In summary, the functional state of the fast twitch skeletal muscle calcium release channel can be characterized by the changes in the kinetics of [3H]ryanodine binding. Different modulators (activators/inhibitors) affect different aspects of ryanodine binding (association/dissociation).

Adenylyl Imidodiphosphate↗

PubMed1989

[3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes.

Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [3H]PN200-110, and the alkaloid, [3H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.

Alkaloids↗

PubMed1989

Subunit composition of the purified dihydropyridine binding protein from skeletal muscle.

The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1989

A procedure for purification of the ryanodine receptor from skeletal muscle.

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636-16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [3H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.

Animals↗

PubMed1989

Functional anatomy of the second visual area (V2) in the macaque.

To study the functional organization of secondary visual cortex (V2) in the primate, 14C-2-deoxy-d-glucose (DG) was injected while macaque monkeys were shown specific visual stimuli. Wherever possible, patterns of DG uptake were compared with the position of dark and light cytochrome oxidase (cytox) stripes (Tootell et al., 1983). Often, the DG effects of 2 different stimuli were compared in the same hemisphere to eliminate ambiguities inherent in between-animal comparisons. Data were obtained from a large number of animals in conjunction with related DG studies in area V1 (primary visual or striate cortex). The following conclusions were reached: (1) in some macaque monkeys, dark cytox stripes were faint or absent. Although this could conceivably be due to poor staining technique, some evidence suggests that the lack of enzyme stripe pattern is real. In all animals, including those that showed poor or no cytox staining evidence for stripes, the functional architecture revealed by the DG was consistently present and robust. (2) Uniform gray stimuli produce a relatively uniform pattern and minimal stimulus-related DG uptake. (3) Eye movements per se produce some uptake in the V2 stripes. (4) Very generalized visual stimulation conditions (e.g., binocular stimulation with a grating of varied orientation and varied spatial frequency) produced a pattern of uptake that is greatest in both sets of dark cytox stripes and lighter in the light cytochrome stripes. (5) In both the DG and cytox results, the V2 "stripes" are more accurately described as stripe-shaped collections of patches. (6) In almost all cases, DG patterns were columnar in shape, extending from white matter to cortical surface. The boundaries of the columns were most sharply defined, and the contrast was highest, in layers 3B/4, becoming slightly more blurry and lower in contrast in other layers. Laminar differences between DG patterns in V2 were almost negligible, compared with the profound laminar differences in macaque V1. (7) There is no DG evidence for, and much against, the possibility of an ocular dominance architecture in V2. (8) There are orientation columns in macaque V2. DG-labeled orientation columns are spaced further apart than those in V1, by a factor of about 1.6, but the columns are not correspondingly wider. (9) Spatially diffuse variations in color produce high uptake confined, at least largely, to the thin cytox stripes. (10) There is evidence for spatially antagonistic color surrounds in color cells in the thin stripes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist.

We demonstrated recently that purified preparations of Gs, the stimulatory G protein of adenylyl cyclase, can stabilize Ca2+ channels in inside-out cardiac ventricle membrane patches stimulated prior to excision by the beta-adrenergic agonist isoprenaline or by the dihydropyridine agonist Bay K 8644 and that such preparations of Gs can restore activity to spontaneously inactivated cardiac Ca2+ channels incorporated into planar lipid bilayers (Yatani, A., Codina, J., Reeves, J.P., Birnbaumer, L., and Brown, A.M. (1987) Science 238, 1288-1292). To test whether these effects represented true stimulation and to further identify the G protein responsible, we incorporated skeletal muscle T-tubule membranes into lipid bilayers and studied the response of their Ca2+ channels to G proteins, specifically Gs, and manipulations known to be specific for Gs. In contrast to cardiac channels, incorporated T-tubule Ca2+ channels exhibit stable average activities over prolonged periods of time (up to 20 min at room temperature), allowing assessment of possible effects of G proteins under steady-state assay conditions. We report that exogenously added human erythrocyte GTP gamma S (guanosine 5'-O-(3-thiotriphosphate]-activated Gs (Gs) or its resolved GTP gamma S-activated alpha subunit (alpha s) stimulate T-tubule Ca2+ channels by factors of 2-3 in the presence of Bay K 8644, and of 10-20 in the absence of Bay K 8644 and that they do so in a manner that is independent of concurrent or previous phosphorylation by cAMP-dependent protein kinase. Activation of purified Gs by cholera toxin increases both its adenylyl cyclase stimulatory and its Ca2+ channel stimulatory effects. Ca2+ channels previously stimulated by the combined actions of Bay K 8644 and cAMP-dependent protein kinase still respond to Gs. We conclude that the responses seen are due to Gs rather than a contaminant, that the effect on Ca2+ channel activity is that of a true stimulation, akin to that on adenylyl cyclase, and show that a given G protein may regulate more than one effector system.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1988

A plasma membrane-enriched preparation from giant barnacle muscle fibers.

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1988

Functional anatomy of macaque striate cortex. I. Ocular dominance, binocular interactions, and baseline conditions.

A series of experiments was carried out using 14C-2-deoxy-d-glucose (DG) in order to examine the functional architecture of macaque striate (primary visual) cortex. This paper describes the results of experiments on uptake during various baseline (or reference) conditions of visual stimulation (described below), and on differences in the functional architecture following monocular versus binocular viewing conditions. In binocular "baseline" experiments, monkeys were stimulated either (1) in the dark, (2) with a diffuse gray screen, or (3) with a very general visual stimulus composed of gratings of varied orientation and spatial frequency. In all of these conditions, DG uptake was found to be topographically uniform within all layers of parafoveal striate cortex. In monocular experiments that were otherwise similar, uptake was topographically uniform within the full extent of the eye dominance strip, in all layers. Certain other visual stimuli produce high uptake in the blobs, and still another set of visual stimuli (including high-spatial-frequency gratings) produce highest uptake between the blobs at parafoveal eccentricities, even in an unanesthetized, unparalyzed monkey. Eye movements per se had no obvious effect on striate DG uptake. Endogenous uptake in the blobs (relative to that in the interblobs) appears higher in the squirrel monkey than in the macaque. The pattern of DG uptake produced by binocular viewing was found to deviate in a number of ways from that expected by linearly summing the component monocular DG patterns. One of the most interesting deviations was an enhancement of the representation of visual field borders between stimuli differing from each other in texture, orientation, direction, etc. This "border enhancement" was confined to striate layers 1-3 (not appearing in any of the striate input layers), and it only appeared following binocular, but not monocular, viewing conditions. The border enhancement may be related to a suppression of DG uptake that occurs during binocular viewing conditions in layers 2 + 3 (and perhaps layers 1 and 4B), but not in layers 4Ca, 4Cb, 5 or 6. Another major class of binocular interaction was a spread of neural activity into the "unstimulated" ocular dominance strips following monocular stimulation. Such an effect was prominent in striate layer 4Ca, but it did not occur in layer 4Cb. This "binocular" spread of DG uptake into the inappropriate eye dominance strip in 4Ca may be related to the appearance of orientation tuning and orientation columns in that layer. No DG effects were seen that depended on the absolute disparity of visual stimuli in macaque striate cortex.

Animals↗

PubMed1988

Functional anatomy of macaque striate cortex. II. Retinotopic organization.

Macaque monkeys were shown retinotopically-specific visual stimuli during 14C-2-deoxy-d-glucose (DG) infusion in a study of the retinotopic organization of primary visual cortex (V1). In the central half of V1, the cortical magnification was found to be greater along the vertical than along the horizontal meridian, and overall magnification factors appeared to be scaled proportionate to brain size across different species. The cortical magnification factor (CMF) was found to reach a maximum of about 15 mm/deg at the representation of the fovea, at a point of acute curvature in the V1-V2 border. We find neither a duplication nor an overrepresentation of the vertical meridian. The magnification factor did not appear to be doubled in a direction perpendicular to the ocular dominance strips; it may not be increased at all. The DG borders in parvorecipient layer 4Cb were found to be as sharp as 140 micron (half-amplitude, half width), corresponding to a visual angle of less than 2' of arc at the eccentricity measured. In other layers (including magnorecipient layer 4Ca), the retinotopic borders are broader. The retinotopic spread of activity is greater when produced by a low-spatial-frequency grating than when produced by a high-spatial-frequency grating. Orientation-specific stimuli produced a pattern of activation that spread further than 1 mm across cortex in some layers. Some DG evidence suggests that the spread of functional activity is greater near the foveal representation than near 5 degrees eccentricity.

Animals↗

PubMed1988

Functional anatomy of macaque striate cortex. III. Color.

Using spatially diffuse stimuli (or sinusoidal gratings of very low spatial frequency), levels of 14C-2-deoxy-d-glucose (DG) uptake produced by color-varying stimuli are much greater than those produced by luminance-varying stimuli in macaque striate cortex. Such a difference in DG results is consistent with previous psychophysical and electrophysiological results from man and monkey. In DG experiments with color-varying gratings of low and middle spatial frequencies, or with spatially diffuse color variations, DG uptake was highest in the cytochrome oxidase blobs, as was also seen with low-spatial-frequency luminance gratings. High-spatial-frequency, color-varying uptake patterns were shifted to cover both blob and interblob regions in a manner similar to that of the patterns obtained with middle-spatial-frequency luminance stimuli. However, in no instance did chromatic gratings produce uptake restricted to the interblob regions, as with the pattern seen with the highest-spatial-frequency luminance gratings. Thus, DG uptake is relatively higher in the interblob regions when comparing luminance with color-varying gratings that are otherwise similar. It was also possible to show DG evidence for receptive-field double-opponency in the upper-layer blobs, but color sensitivity in layer 4Cb appears single-opponent. The DG results suggest that color sensitivity is also high in the lower-layer (layers 5 + 6) blobs, and that many layer 5 receptive fields are double-opponent. Striate layers 4Ca and 4B-appeared color-insensitive in a wide variety of DG tests; this supports the idea of a color-insensitive stream running from the magnocellular LGN layers through striate layers 4Ca and 4B to extrastriate areas MT and V3. There was also a major effect due to wavelength: long and short wavelengths produced much more uptake than did middle wavelengths, even when all colors were equated for luminance and saturation. No variation with eccentricity was seen in cortical color sensitivity, at least between 0 degrees and 10 degrees.

Animals↗

PubMed1988

Functional anatomy of macaque striate cortex. IV. Contrast and magno-parvo streams.

Macaque monkeys were shown achromatic gratings of various contrasts during 14C-2-deoxy-d-glucose (DG) infusion in order to measure the contrast sensitivity of different subdivisions of primary visual cortex. DG uptake is essentially saturated at stimulus contrasts of 50% and above, although the saturation contrast varies with layer and with different criteria. Following visual stimulation with gratings of 8% contrast, stimulus-driven uptake was relatively high in striate layer 4Ca (which receives primary input from the magnocellular LGN layers), but was absent in layer 4Cb (which receives primary input from the parvocellular layers). In this same (magnocellular-specific) stimulation condition, striate layers 4B, 4Ca, and 6 showed strong stimulus-induced DG uptake, and layers 2, 3, 4A, and 5 showed only light or negligible uptake. By comparison to other cases that were shown stimuli of systematically higher contrast, and to a wide variety of DG cases shown very different stimuli, it is evident that information derived from the magnocellular and parvocellular layers in the LGN remains partially, or largely, segregated in its passage through striate cortex, and projects in a still somewhat segregated fashion to different extrastriate areas. The sum of all available evidence suggests that the magnocellular information projects strongly through striate layers 4Ca, 4B, and 6, with moderate input into the blobs in layers 2 + 3, and to blob-aligned portions of layer 4A. Parvocellular-dominated regions of striate cortex include both the blob and interblob portions of layers 2 + 3, 4A, 4Cb, and 5. Because the major striate input to V2 arrives from striate layers 2 + 3, and because the major striate input to MT originates in layer 4B and 6, it appears that area V2 receives information derived largely from the parvocellular LGN layers, and that area MT receives information derived mainly from the magnocellular layers.

Animals↗

PubMed1988

Functional anatomy of macaque striate cortex. V. Spatial frequency.

When macaque monkeys view achromatic, sinusoidal gratings of a single spatial frequency, the pattern of 14C-2-deoxy-d-glucose (DG) uptake produced by the gratings is shown to depend on the spatial frequency chosen. When a relatively high (5-7 cycles/deg) spatial frequency is shown binocularly at systematically varied orientations, uptake in parafoveal striate cortex is highest between the cytochrome oxidase blobs (that is, in the interblobs) in layers 1, 2, and 3. In layers 4B, 5, and 6, where the cytochrome oxidase blobs are faint or absent, DG uptake is highest in a periodic pattern that lies in register with the interblobs of layers 2 + 3. When the grating is, instead, of relatively low (1-1.5 cycles/deg) spatial frequency, DG uptake is highest in the blobs, in the blob-aligned portions of layers 1-4B, and in the lower-layer blobs as well. These variations in DG topography are confirmed in stimulus comparisons within a single hemisphere. Presumably, this shift in functional topography within the extra-granular layer is the primate homolog of "spatial frequency columns" shown earlier in the cat (Tootell et al., 1981; Silverman, 1984). In the well-differentiated architecture of primate striate cortex, laminar differences produced by high- versus low-spatial-frequency gratings are visible as well. Gratings of very high spatial frequency produce much higher uptake in 4Cb (which receives input from the parvocellular LGN layers) than in 4Ca (which gets its input from the magnocellular LGN layers). Gratings of low spatial frequency produce the converse result. Presumably, cells in the magnocellular LGN layers and/or in the magnocellular-dominated layer 4Ca have lower average spatial frequency tuning (larger receptive fields) than their counterparts in the parvocellular LGN and/or in striate layer 4Cb. The DG patterns produced by various spatial frequencies also vary with eccentricity, in a manner consistent with known, eccentricity-dependent variations of receptive-field size and spatial frequency tuning. Thus, gratings of a "middle"-spatial-frequency range (4-5 cycles/deg) produce high uptake in the blobs near the foveal representation and high uptake in the interblobs at more peripheral eccentricities, including 5 degrees. This shift in DG topography also includes the transition zone near 3 degrees, where the level of stimulus-driven uptake is as high in the blob regions as it is in interblob regions. Variations in uptake between layers 4Ca and 4Cb, as a function of eccentricity, shift in parallel with the changes in the upper-layer topography.

Animals↗

PubMed1987

Low affinity binding sites for 1,4-dihydropyridines in mitochondria and in guinea pig ventricular membranes.

In this paper, we describe the occurrence of both high and low affinity sites for dihydropyridines in crude membrane preparations from guinea pig ventricular tissue. The physiological significance of the low affinity site (apparent dissociation constant = 76 +/- 9 nM) is not currently known; it has, however, a binding capacity which was 300-1000 times that of the high affinity site and was resistant to heat denaturation. The magnitude of the binding to the low affinity site was affected by both the ionic strength of the medium and by the presence of divalent ions. Both unlabeled nitrendipine and nimodipine inhibited [3H]nitrendipine binding at both sites, but verapamil and diltiazem only affected binding at the high affinity site. We also characterized, both kinetically and by equilibrium binding, a low affinity, heat-stable nitrendipine binding site in purified mitochondria. The Bmax for this site was also dependent on ionic strength. This suggests the possibility that the low affinity site in crude membranes is due to mitochondrial contaminants and hence not directly related to voltage-dependent calcium channels.

Animals↗

PubMed1987

Toxins that affect voltage-dependent calcium channels.

At this time, there are five potential candidates for calcium channel specific toxins. All five of these toxins appear to affect the function of voltage-dependent calcium channels. Atrotoxin, beta-leptinotarsin-h and maitotoxin activate channels, whereas both taicatoxin and omega-conotoxin are inhibitors. Neither maitotoxin nor omega-conotoxin alters the binding of dihydropyridines to membranes derived from the cells upon which the toxins exert their effects. In contrast, both atrotoxin and taicatoxin inhibit the binding of dihydropyridines to ventricular membranes. It is not currently known whether beta-leptinotarsin-h affects the binding. The effects of maitotoxin and atrotoxin are blocked by dihydropyridines and verapamil. Direct binding studies with radiolabeled toxins have been performed only with omega-conotoxin, and the binding site density for this toxin appears to be at least one order of magnitude greater than the density of dihydropyridine binding sites in synaptosomes. Studies to examine the third and fourth criteria which we have listed (i.e. that the effects are not via a second messenger, or an enzyme activity) have not been reported for either beta-leptinotarsin-h or omega-conotoxin. Atrotoxin and taicatoxin, added outside a patch pipette, have no effects on calcium channels within the patch and are, therefore, probably not affecting calcium channels via a second messenger. Maitotoxin, however, affects the formation of inositol phosphates and, hence, could be affecting the channel indirectly. The fractions containing the toxic components atrotoxin and taicatoxin have no phospholipase or protease activity, and this is presumably true also for omega-conotoxin since it has been purified to homogeneity. Although all of the toxins have the potential to be important tools with which to study calcium channel structure and function, a number of experiments remain to be done in order to establish conclusively that these five toxins bind specifically to the voltage-dependent calcium channel. In conclusion, we would like to briefly mention why so much effort is being devoted to the search for these calcium channel specific toxins. Such a toxin would provide a very valuable tool in the study of calcium channels for a number of reasons. First, the toxin would be another ligand for the channel and, as such, would provide an alternative to organic ligands such as the dihydropyridines which are lipophilic and, in many tissues, have more than one binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1987

A comparison between the binding and electrophysiological effects of dihydropyridines on cardiac membranes.

To investigate the mechanism of action of dihydropyridines on calcium channels, both receptor binding and the electrophysiological effects of optically pure enantiomers of Bay k 8644, Sandoz 202-791, nimodipine, and PN200-110 were studied in a guinea pig ventricular preparation. The radioligand binding studies are consistent with other reports that the inhibitory and excitatory dihydropyridines share a single binding site. The patch clamp method was used for recording whole cell calcium currents. (-)-Bay k 8644 and (+)-202-791 produced voltage-dependent increases in the calcium currents. The magnitude of the effect was dependent on the holding potential. At holding potentials between -40 and -90 mV these activators increased calcium currents in a concentration-dependent manner with EC50 values of 25 nM and 80 nM, respectively. The inhibitors (+)-Bay k 8644, (-)-202-791, (+)- and (-)-nimodipine, and (+)-PN200-110 blocked the calcium currents with potencies that depended upon holding potential. The IC50 values for these enantiomers measured at a holding potential of -80 mV were, respectively, 8000, 200, 2000, 450, and 400 nM, and IC50 values measured at a holding potential of -30 mV were 26, 1.0, 52, 4.0, and 4.5 nM. The dissociation constants calculated for some dihydropyridines are similar to the Kd values determined by radioligand binding. However, for other dihydropyridines, large discrepancies between the concentrations giving rise to half-maximal electrophysiological effects and the Kd values from binding studies could not be reconciled by voltage-dependent binding alone. We suggest that each dihydropyridine also produces unique effects on the voltage-dependent gating of calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1987

Dihydropyridine binding and calcium channel function in clonal rat adrenal medullary tumor cells.

We correlated the binding of the dihydropyridines, nitrendipine and PN200-110, with their pharmacological actions on voltage-dependent membrane calcium channels. Binding was studied in clonal rat adrenal medullary cells (PC12) and in plasma membranes prepared from them. Calcium currents were studied using whole cell and single channel patch clamp methods. For both [3H]-(+/-)-nitrendipine and [3H]-(+)-PN200-110, high affinity binding sites with dissociation constants of 0.6 and 0.04 nM, respectively, were identified both in membrane fragments and in intact cells. In crude membrane preparations a low affinity nitrendipine-binding site was also found. The dissociation constant for binding at this site was affected by ionic strength and the presence of divalent cations. In 500 mM KCI, 0.1 mM CaCl2, 50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), the KD is about 70 nM. The number of high affinity binding sites for dihydropyridines was between 30 and 100 fmol/mg of protein while the number of low affinity sites was between 30 and 70 pmol/mg of protein. In whole cells the measured number of high affinity sites was between 2000 and 4000/cell and, by extrapolation from the membrane preparation, the low affinity sites correspond to several million sites per cell. The electrophysiological effects of both of the dihydropyridines on Ca2+ currents were voltage dependent. When nitrendipine was applied, a small increase in calcium current occurred and this was followed by a decrease. The inhibitory effect was more pronounced at depolarized membrane holding potentials and was relieved by hyperpolarizing the membrane, whereas the stimulatory effect was pronounced at negative membrane holding potentials. In 10 nM nitrendipine these effects were also observed in single channels; they were not due to changes in channel conductance or dwell times in the open state but, rather, were due to changes in the probability of opening. The half-maximal inhibitory concentration (IC50) for nitrendipine was 67 nM and the IC50 for the effect of (+)-PN200-110 was 9 nM using protocols which favored the depolarized state of the channel. No excitatory effect was seen. The IC50 from electrophysiological estimates is higher than the KD for the high affinity binding site for both compounds. Using a simple model of voltage-dependent binding, we could not account for the difference. The number of functional channels calculated from the relation between whole cell and single channel calcium currents and the probability of opening was in good agreement with the number of high affinity sites calculated from the binding studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Gland Neoplasms↗

PubMed1986

Diphenylhydantoin blocks cardiac calcium channels and binds to the dihydropyridine receptor.

Diphenylhydantoin was studied for its effects on Ca currents in single isolated guinea pig ventricular cells. The whole-cell patch-clamp technique was used, and Ca currents were studied after suppressing Na and K currents. At low frequencies (0.1 Hz) and negative holding potentials (-50 mV), diphenylhydantoin produced a concentration-dependent decrease in Ca currents without any significant change in the current-voltage relations. Half blocking effect occurred at 2 X 10(-4) M. The effects of diphenylhydantoin on Ca currents were dependent upon the holding potential. Inactivation curves for Ca currents were shifted to more negative potentials by the drug. The recovery of Ca currents from inactivation was prolonged by diphenylhydantoin, and the repriming of the current displayed an additional component, attributed to slow release of the drug from the channels. The voltage-dependent block was attributed to preferred binding by the inactivated channel state. Diphenylhydantoin also blocked specific [3H]-nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of [3H]-nitrendipine binding by diphenylhydantoin was competitive. Diphenylhydantoin also blocks cardiac Na channels in a voltage-dependent manner. We suggest that diphenylhydantoin binding sites exist on both Ca and Na channels.

Animals↗

PubMed1985

Atrotoxin: a specific agonist for calcium currents in heart.

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.

Action Potentials↗