Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

1989 ◽  
Vol 67 (5) ◽  
pp. 506-514 ◽  
Author(s):  
X. Y. Wei ◽  
A. Rutledge ◽  
Q. Zhong ◽  
J. Ferrante ◽  
D. J. Triggle
Keyword(s):  
Calcium Channel ◽  
Intact Cell ◽  
Neural Retina ◽  
Bay K 8644 ◽  
Single Type ◽  
Channel Blockers ◽  
Binding Affinities ◽  
High Affinity ◽  
Voltage Dependent ◽  
High Affinity Binding Site

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site. [3H]PN 200-110 bound with higher affinity (KD = 7.09 ± 0.90 × 10−11 M) than did [3H]nitrendipine (KD = 4.10 ± 0.92 × 10−10 M), but the Bmax values were similar for the two ligands (98.9 ± 4.1 and 99.4 ± 6.58 fmol/mg protein, respectively). The discrepancy between binding and pharmacologic activities of the antagonist ligands does not appear to be due to the presence of 1,4-dihydropyridine-insensitive Ca2+ channels, but may relate to the inability of these agents to access a high affinity inactivated state in the intact cell or to the presence of discrete categories of binding sites. This marked discrepancy between affinities does not exist for the activator ligands studied. This study confirms the presence of voltage-dependent 1,4-dihydropyridine sensitive Ca2+ channels in chick neural retina cells.Key words: Ca2+ channels, 1,4-dihydropyridines, chick neural retina, retinal neurons.

scholarly journals Bio-inspired voltage-dependent calcium channel blockers

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Tingting Yang ◽  
Lin-Ling He ◽  
Ming Chen ◽  
Kun Fang ◽  
Henry M. Colecraft
Keyword(s):  
Calcium Channel ◽  
Calcium Channel Blockers ◽  
Channel Blockers ◽  
Voltage Dependent Calcium Channel ◽  
Voltage Dependent

scholarly journals Photoaffinity labelling of the cardiac calcium channel. (−)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand

1987 ◽  
Vol 243 (1) ◽  
pp. 127-135 ◽  
Author(s):  
D R Ferry ◽  
A Goll ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dimethyl Ester ◽  
High Capacity ◽  
Bay K 8644 ◽  
Channel Blockers ◽  
Guinea Pig Heart ◽  
Affinity Ligand ◽  
Heart Membranes

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

Calcium dependence of effects of endothelin on rat mesenteric microvessels

1991 ◽  
Vol 69 (6) ◽  
pp. 798-804 ◽  
Author(s):  
Li Yuan Deng ◽  
Ernesto L. Schiffrin
Keyword(s):  
Calcium Channel ◽  
Channel Blocker ◽  
Extracellular Fluid ◽  
Channel Blockers ◽  
Active Tension ◽  
Calcium Dependence ◽  
Rat Mesentery ◽  
Resistance Vessels ◽  
Voltage Dependent ◽  
Mesenteric Microvessels

We investigated the calcium dependence of the effects of endothelin (ET) on resistance vessels (less than 300 μm lumen diameter) from the mesenteric vascular bed of the rat, mounted on a wire myograph. ET-1 induced a potent sustained contraction with an ED50 of 12 nmol/L. The response to ET-3 and big ET at the maximum concentrations used (100 nmol/L) was less than 40% of that to ET-1, with an estimated ED50 of 45 nmol/L. Relaxation of the ET-1-induced contraction was slow, and resulted in a reduction of the maximum response to a second challenge with ET-1 to 60% of the initial contraction after 3 h. Long-lasting tachyphylaxis to arginine vasopressin (AVP) induced contraction also occurred. The response to 100 nmol/L ET-1 produced an active tension 88% greater than that induced by 124 mmol/L KCl, and similar to that produced by norepinephrine and AVP. The response to 100 nmol/L ET-1 in the absence of calcium + 1 mmol/L EGTA in the medium for 30 min resulted in a maximum contraction of 43% of the response in the presence of calcium, followed by a faster relaxation rate. The addition of calcium produced a further contraction, and stimulation with 100 nmol/L ET-1 at this point did not result in further response. The calcium channel blocker nitrendipine in concentrations of 1–10 μmol produced increasing reductions of the responses to 100 nmol/L ET-1 to 35% at the higher concentration. Nitrendipine (3 μmol/L) partially blocked the response to calcium after ET-1 was added in the absence of calcium. We conclude that resistance microvessels of the rat mesentery are more sensitive to ET-1 than ET-3 or big ET, which were also much less potent. These microvessels have a sensitivity to AVP 20 times greater than that to ET-1. The response to ET-1 may be mediated in part by the release of intracellular calcium, but the influx of calcium from the extracellular fluid through plasma membrane voltage-dependent channels is necessary for completing approximately 60% of the initial contraction and for the persistence of a sustained response.Key words: mesenteric arterioles, vasopressin, norepinephrine, nitrendipine, calcium channel blockers.

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